Effect of Gibberellin and Heat Shock on the Lipid Composition of Endoplasmic Reticulum in Barley Aleurone Layers

The heat-shock responses of barley (Hordeum vulgare L. cv Hi- malaya) aleurone layers incubated with or without gibberellic acid (GA3) were compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat shock blocked the synthesis and secretion of secretory proteins from GA3-treated layers but not untreated layers. This suppression of secretory protein synthesis has been correlated with changes in endoplasmic reticulum (ER) membranes (F.C. Belanger, M. R. Brodl, T.-h.D. Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358; L. Sticher, A.K. Biswas, D.S. Bush, R.L. Jones [1990] Plant Physiol 92: 506-513). Our secretion data suggested that the ER membranes of aleurone layers incubated without GA3 may be more heat shock tolerant. To investigate this, the lipid profiles of membrane extracts in aleurone layers labeled with [14C]glycerol were examined. Heat shock markedly increased [14C]glycerol incorporation into phosphatidylcholine (PC), and gas chromatography revealed an increase in the amount of saturated fatty acids associated with thin layer chromatography-purified PC in GA3-treated layers. In contrast, aleurone layers incubated without GA3 at normal temperature contained PC-associated fatty acids with a greater degree of saturation than GA3-treated layers. Heat shock modestly increased the degree of fatty acid saturation in untreated aleurone layers. This same trend was noted in fatty acids isolated from ER membranes purified by continuous sucrose density centrifugation. We propose that increased fatty acid saturation may help sustain ER membrane function in heat-shocked aleurone layers incubated in the absence of GA3.     

Gibberellic Acid Regulates the Level of a BiP Cognate in the Endoplasmic Reticulum of Barley Aleurone Cells

The isolation of a 70-kilodalton protein from barley (Hordeum vulgare L.) aleurone layers that cross-reacts with an antibody against yeast binding protein (BiP) is reported. Endoplasmic reticulum isolated from aleurone layers treated with gibberellic acid contain much higher levels of the BiP cognate than do membranes isolated from layers treated with abscisic acid.     

Heat Shock Causes Selective Destabilization of Secretory Protein mRNAs in Barley Aleurone Cells

The aleurone layer of GA₃-stimulated barley (Hordeum vulgare L., cv Himalaya) grains is normally devoted to the synthesis and secretion of hydrolytic enzymes. Heat shock, however, suppresses the synthesis of the main hydrolytic enzyme, α-amylase, by destabilizing its otherwise highly stable mRNA (FC Belanger, MR Brodl, T-hD Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358). In this paper we document that heat shock causes the suppression of the synthesis of some normal cellular proteins, while the synthesis of other normal cellular proteins is unaffected by heat shock. There are two major isozymic forms of α-amylase encoded by distinct mRNAs. The mRNA levels for both isozymic forms and the mRNA levels of two other secretory proteins, a protease and an endochitinase, were markedly reduced during heat shock. However, the levels of actin and β-tubulin mRNAs, both nonsecretory proteins, were not diminished during heat shock. In addition, the levels of three other mRNA species detected by a set of unidentified cDNA clones (the sequence of one shows that it lacks a signal sequence) remained unchanged during heat shock. These data indicate that there are two classes of normal cellular protein mRNAs with regard to the effect of heat shock upon their persistence in the cell, and suggest that the distinction between them is whether or not they encode secretory proteins.     

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome,Protein Secretion,and Endoplasmic Reticulum Stress

The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.Plant proteins that are secreted to the apoplast have important functions in signaling, defense, and cell regulation. The classical protein secretory pathway is less characterized in plants than in mammals or yeast but is of growing interest due to the potential of plant systems as hosts for the production of recombinant proteins (Erlendsson et al., 2010; De Wilde et al., 2013). Plant secretomics, therefore, is a rapidly expanding area applied to gain further insight into these processes (Agrawal et al., 2010; Alexandersson et al., 2013). Many secretory proteins contain putative N-glycosylation sites, and the identification and characterization of these sites is an important element in secretomics analysis. However, to date, only a few plant glycoproteomes have been described (Fitchette et al., 2007; Minic et al., 2007; Palmisano et al., 2010; Melo-Braga et al., 2012; Zhang et al., 2012; Thannhauser et al., 2013).The cereal aleurone layer is of major importance due to its central role in grain germination. Previous proteomic studies have been reported for aleurone layers dissected from mature (Finnie and Svensson, 2003) and germinating (Bønsager et al., 2007) barley (Hordeum vulgare) or developing (Tasleem-Tahir et al., 2011) and mature (Laubin et al., 2008; Jerkovic et al., 2010; Meziani et al., 2012) wheat (Triticum aestivum) grains. The in vitro culture of isolated aleurone layers was developed by Chrispeels and Varner (1967). Since then, this system has become an excellent tool for the study of germination signaling in response to phytohormones (Bush et al., 1986; Jones and Jacobsen, 1991; Bethke et al., 1997; Ishibashi et al., 2012). More recently, it has been adopted as a unique system for the analysis of plant secretory proteins (Hägglund et al., 2010; Finnie et al., 2011). The addition of GA₃ to the isolated aleurone layers induces the synthesis and secretion of hydrolytic enzymes. In the in vitro system, these accumulate in the incubation buffer, facilitating their identification and characterization using proteomics techniques. Thus, numerous secreted proteins with roles in the hydrolysis of starch, cell wall polysaccharides, and proteins could be identified (Finnie et al., 2011). Furthermore, several of the proteins were also detected in intracellular extracts from the same aleurone layers, presumably prior to their release. Many of the proteins appeared in multiple forms on two-dimensional (2D) gels, and often with higher M_r than expected, suggesting the presence of posttranslational modifications (PTMs; Finnie et al., 2011). Bak-Jensen et al. (2007) and Finnie et al. (2011) observed a highly complex pattern of α-amylase-containing spots on 2D gels, which originated from only two α-AMYLASE2 (AMY2) and two AMY1 gene products from a total of 10 genes, probably reflecting multiple forms due to PTMs.In eukaryotic cells, proteins synthesized in the endoplasmic reticulum (ER) must be correctly folded and assembled before continuing in the secretory pathway. Perturbations of redox state, calcium regulation, Glc deprivation, and viral infection can lead to ER stress, triggered by the accumulation of unfolded and misfolded proteins in the ER lumen. This provokes a triple response from the cell, consisting of an up-regulation of chaperones and vesicle trafficking, a down-regulation of genes encoding secretory proteins, and an up-regulation of proteins involved in endoplasmic reticulum-associated protein degradation (ERAD). In plants, the molecular mechanisms underlying ER stress in plants have yet to be fully resolved (Martínez and Chrispeels, 2003; Nagashima et al., 2011; Moreno et al., 2012).Tunicamycin (TN), an inhibitor of GlcNAc phosphotransferase, which catalyzes the first step in glycoprotein synthesis, has been used to induce ER stress by causing the accumulation of unfolded proteins in the ER lumen (Noh et al., 2003; Kamauchi et al., 2005; Reis et al., 2011). If unfolded proteins are not removed, the prolonged stress will induce programmed cell death. Links between ER stress and apoptosis have been reported in response to TN treatment in mammalian cells, whereas in plants, this correlation has been suggested, but the pathways of signal transduction remain unknown (Kamauchi et al., 2005; Reis et al., 2011).In all plant tissues, heat shock (HS) induces the synthesis of a variety of heat shock proteins (HSPs), which are responsible for protein refolding under stress conditions (Craig et al., 1994) and translocation and degradation in a broad array of normal cellular processes (Bond and Schlesinger, 1986; Spiess et al., 1999). In the barley aleurone layer, HS selectively suppresses the synthesis of secretory proteins, including α-amylase, due to the selective destabilization of secretory protein mRNA (Belanger et al., 1986; Brodl and Ho, 1991). However, an acclimation effect has been described in aleurone cells after prolonged incubation at warm temperatures, resulting in a resumption of the protein secretory machinery (Shaw and Brodl, 2003). The connection between heat stress response and ER stress has been well established in mammals and yeast, but scarce information is available in plants (Denecke et al., 1995).Over the last years, the dual role of reactive oxygen species (ROS) has been established in plants: at higher concentrations, ROS act as toxic molecules damaging cellular macromolecules, eventually causing cell death, but at lower concentrations, ROS seem to be necessary for seed germination and seedling growth by controlling the cellular redox status, regulating growth and protecting against pathogens (Bailly, 2004; Bailly et al., 2008). In the barley aleurone layer, GA₃ perceived at the plasma membrane induces ROS generation as a by-product from intense lipid metabolism, and the redox regulation of the GA₃-induced response has been proposed (Maya-Ampudia and Bernal-Lugo, 2006). This suggests that during the secretory function of the tissue, moderate levels of ROS may be acting as cellular messengers. In aleurone cells, ROS, especially hydrogen peroxide (H₂O₂), are involved in the process of programmed cell death, but the molecular mechanisms remain unclear (Bethke and Jones, 2001; Ishibashi et al., 2012).Until now, none of the protein components of the ER stress pathways have been identified in barley; also, little is known about the glycosylation of barley proteins. In this work, numerous N-glycoslation sites are identified, and the effect of perturbing N-glycosylation and the secretory pathway by TN and HS treatments is analyzed in GA₃-induced barley aleurone layers.     

alpha-Amylase Isoforms are Posttranslationally Modified in the Endomembrane System of the Barley Aleurone Layer

The subcellular site of the posttranslational modification of α-amylase was investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya). Aleurone layers of Himalaya barley synthesize and secrete two groups of α-amylase isoforms, referred to as low-isoelectric point (low-pl) or HAMY1 and high-pl or HAMY2, when incubated in gibberellic acid and CaCl₂. Whereas homogenates of aleurone layers contain four isoforms of HAMY1 with pls 4.90, 4.72, 4.64, and 4.56, incubation media contain predominantly isoforms 4.72 and 4.56. Microsomal membranes isolated from aleurone layers contain all four isoforms of HAMY1. Microsomal membranes can be resolved into two peaks by isopycnic density gradient centrifugation: a peak of heavy membranes with endoplasmic reticulum and Golgi apparatus (GApp) marker enzyme activities and a peak of light membranes with characteristics of the GApp. The heavy membranes contain proportionally more HAMY1 pl 4.90 and 4.64 isoforms, whereas light membranes contain a higher proportion of pl 4.72 and 4.56 isoforms. Experiments with the ionophore monensin show that membranes of the GApp as well as the endoplasmic reticulum are involved in the posttranslational modification of HAMY1 isoforms. Monensin inhibits the secretion of α-amylase and causes the enzyme to accumulate within the cell. Precursor forms of HAMY1 accumulate in light membranes isolated from monensin-treated aleurone layers indicating that the GApp is involved in the conversion of the precursor to the secreted forms of the enzyme.     

Hypochlorite Disinfection Influences the GA(3)-Induced Synthesis of alpha-Amylase Isozymes by Isolated Barley Aleurone Layers

Aleurone layers isolated from half-seeds of Himalaya barley (Hordeum vulgare cv Himalaya) disinfected in hypochlorite solutions containing 1.0% available chlorine synthesized significantly less α-amylase in response to gibberellic acid than layers derived from half-seeds disinfected in 0.1% hypochlorite. This effect of hypochlorite involved neither a differential decrease in the synthesis of group A or B α-amylase isozymes nor a general decrease in α-amylase synthesis attributable to fewer viable aleurone cells in layers from half-seeds disinfected with 1% hypochlorite. Our results emphasize the need to evaluate the potential effects of routine disinfection procedures used in physiological and biochemical studies.     

Effect of Ethylene on the Release of alpha-Amylase through Cell Walls of Barley Aleurone Layers

A large portion of the gibberellic acid (GA₃)-induced α-amylase in isolated aleurone layers is transported into the incubation medium. In the presence of GA₃ and ethylene, an even larger portion of the enzyme is found in the medium. Employing an acid washing technique developed by Varner and Mense (Plant Physiol 1972 49:187-189), it was observed that ethylene significantly reduces the amount of α-amylase trapped by the thick cell walls of aleurone layers. However, the amount of enzyme remaining in the cell (within the boundary of plasma membrane) is not affected by ethylene. Ethylene has no observable effect on membrane formation as measured by the incorporation of [³²P]orthophosphate into phospholipids. Because of these observations it is suggested that ethylene enhances the release of α-amylase, i.e. transport of α-amylase across cell walls, but not the secretion of α-amylase, i.e. transport of α-amylase past the barrier of plasma membrane. The possible mechanism of this ethylene effect is discussed.     

过量表达内质网小分子热激蛋白增强转基因番茄果实的耐冷藏性

利用番茄内质网小分子热激蛋白(ERsHSP)特异性抗体,对番茄果实蛋白进行Western分析,以测定低温冷藏下番茄果实中ERsHSP的表达量,并测定果实硬度、腐烂度和失重率等指标,以比较中蔬4号转ERsHSP基因番茄和未转基因番茄果实在4℃低温下的耐冷藏性.结果表明:在4℃冷藏30 d期间,转基因番茄果实具有较高的ERsHSP表达水平,而未转基因番茄果实中没有ERsHSP的表达;相对于未转基因番茄果实,转基因番茄果实冷害症状轻,并具有较高的果实硬度(平均值为2.84 kg/cm2)、较低的果实腐烂度(平均值为21.03%)和失重率(平均值为6.33%).     

Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro

Posttranslational modifications that give rise to multiple forms of α-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley α-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). α-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and α-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic α-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley α-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify α-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of α-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.     

Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA library was constructed with the heat shocked tomato (Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp ) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in flower and the cold inducible character of mitochondria shsp gene in leaf were discussed.      

番茄线粒体和内质网小分子热激蛋白基因的分子克隆

以热激处理的番茄（Lycopersicon esculentum Mill．）花为实验材料,构建了cDNA库,运用ＲＴ－ＰＣＲ方法克隆番茄粒体和内质网小分子热激蛋白cDNA,利用这两个保守区片段为探针,筛选cDNA库,获得线粒体和内质网小分子热激蛋白全序列cDNA。;通过分析线粒体和内质网小分子热激蛋白基因对温度的反应,发现小分子热激蛋白基因在番茄花中的热激应答温度低于它们在叶片中的热激应答温度,并且番茄叶片中的线粒体小分子热激蛋白基因还具有低温应答特性。对线粒体和内质网小分子热激蛋白基因的分子结构特点,小分子热激蛋白基因在番茄花中的特别热激应答温度的调控机理以及线粒体小分子热激蛋白的基因在中片中的低温度应答成因进行了讨论。     

Presence of Serine Enzymes in Endoplasmic Reticulum of Spinach Callus

Two serine enzymes were detected in microsomes of spinach callusby labeling with [³H]-di-isopropyl phosphorofluoridate (DFP)and examination by SDS-polyacrylamide gel electrophoresis. Thecontents of the larger (mol wt 44,000) and the smaller (molwt 39,000) DFP-binding protein (proteins which have a DFP-reactivesite) were maximum at the third and the second week after cellinoculation, respectively. The positions of both proteins onthe continuous sucrose gradient coincided with that of NADH-cytochromec reductase, a marker enzyme of the endoplasmic reticulum. Thesmaller protein was released from microsomes treated with 0.05%deoxycholate. The larger protein was solubilized with 0.5% cholate,but not with 0.05% deoxycholate, and the apparent molecularweight of the solubilized protein was about 90,000 in 0.5% cholateon Sephacryl S-200 column. [³H]-DFP-binding with the largerprotein was strongly inhibited by DFP, phenylmethylsulfonylfluoride, N-p-tosyl-L-lysine chloromethyl ketone and L-1-tosylamide-2-phenylethylchloromethyl ketone, and slightly by p-chloromercuric benzoateand o-phenanthroline. DFP binding with the smaller one was stronglyinhibited by DFP and PMSF, but not by other reagents. (Received February 12, 1985; Accepted May 27, 1985)     

The Formation of Endoplasmic Reticulum in the Aleurone Cells of Germinating Wheat: an Ultrastructural Study

In the aleurone cells of the quiescent wheat grain endoplasmicreticulum was sparse and present as short profiles. During germinationlong profiles of endoplasmic reticulum developed near to thenuclear membrane and in close association with plastids. Muchof this development occurred during the first 2 d of germination,but further development and stacking of the membranes appearedto take place after this time. The development of the long profileswas independent of the presence of the embryo and thereforeof control by gibberellic acid. On the contrary, after the secondday of germination gibberellic acid produced a decline in theamount of endoplasmic reticulum associated with vesiculationof the reticulum profiles. The results of this ultrastructuralstudy are discussed in the context of available informationon the biosynthesis of phospholipids in aleurone tissue. Someaspects of our results are at variance with those of otherswho have studied the aleurone tissue of barley. These differencesare discussed and suggestions for their resolution are made.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin

HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.     

Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

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     

Roles of the Mitochondria and Endoplasmic Reticulum in Calcium Elevation during Osmotic Shock in Adrenocortical Cells

Steroid hormones participate in various metabolic processes, and dysfunction of the adrenocortical system leads to numerous pathologies in humans. One of the factors that can influence the secretory properties of adrenocorticocytes is changes in the cell volume observed during osmotic shock. In our study, we tested the hypothesis that osmotic stress modifies intracellular Ca²⁺ signalling and in such a way can influence the secretion of steroids by adrenocorticocytes. The effects of hyperosmotic stress on the cytosolic Ca²⁺ concentration ([Ca] _i ) in cultured adrenocortical cells from the zona fasciculata of the rat adrenals were investigated using the indicator fura-2 technique. Our experiments have shown that exposure of the cells to a hyperosmotic solution caused a decrease in the cell volume, as well as a reversible rise in the [Ca] _i . Calcium-free media partly eliminated [Ca] _i responses. Pretreatment of the cells with thapsigargin or CCCP (blockers of internal calcium stores) significantly decreased the magnitude of responses induced by osmotic stress. These findings indicate that osmotic shock causes an increase in the [Ca] _i in adrenocortical cells, mostly due to depletion of the intracellular stores, and may in such a way stimulate steroidogenesis.