Gibberellic Acid Induces Vacuolar Acidification in Barley Aleurone

The roles of gibberellic acid (GA3) and abscisic acid (ABA) in the regulation of vacuolar pH (pHv) in aleurone cells of barley were investigated using the pH-sensitive fluorescent dye 2[prime],7[prime]-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). BCECF accumulated in vacuoles of aleurone cells, but sequestration of the dye did not affect its sensitivity to pH. BCECF-loaded aleurone cells retained their ability to respond to both GA3 and ABA. The pHv of freshly isolated aleurone cells is 6.6, but after incubation in GA3, the pHv fell to 5.8. The pHv of cells not incubated in hormones or in the presence of ABA showed little or no acidification. The aleurone tonoplast contains both vacuolar ATPase and vacuolar pyrophosphatase, but the levels of pump proteins were not affected by incubation in the presence or absence of hormones. We conclude that GA3 affects the pHv in aleurone cells by altering the activities of tonoplast H+ pumps but not the amounts of pump proteins.     

Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

Gibberellic Acid-enhanced Release of beta-1,3-Glucanase from Barley Aleurone Cells

A β-1, 3-glucanase of barley (Hordeum vulgare) aleurone cells accumulates when half-seeds are imbibed on water, and accumulation continues when the aleurone layers are incubated in buffer solution. The release of the enzyme is a gibberellic acid-dependent process, however. Although gibberellic acid stimulates glucanase release, it does not markedly affect the total amount of glucanase obtained from these cells when compared with water controls. β-1, 3-Glucanase release from aleurone cells is a function of gibberellic acid concentration and commences after a 4-hour lag period. Processes occurring during this lag period are also dependent upon gibberellic acid concentration. Removal of gibberellic acid from the incubation medium at the end of the lag period, however, does not affect subsequent release of glucanase. The release of glucanase from aleurone cells is an active process with a Q₁₀ greater than 3. Inhibitors of respiration and protein and RNA synthesis effectively inhibit the formation and release of glucanase. It is concluded that gibberellic acid functions primarily to enhance glucanase release rather than its formation.     

Fine Structural Changes in Barley Aleurone Cells During Gibberellic Acid-induced Enzyme Secretion

The fine structure of barley aleurone cells was studied in theenzyme secretion phase. An ultrastructural feature of this phaseis the formation of stacked rough endoplasmic reticulum (rER),for such a structure was never found in cells during the enzymesynthesis phase. Other structural features frequently observedin the secretion phase were amoeboid-shaped nuclei, the stackedrER wound round the nucleus and mitochondria, and a stream ofthe stacked rER directed to the plasmamembrane. Hordeum vulgare L, barley, aleurone cells, enzyme secretion, gibberellic acid     

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone

Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca²⁺- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca²⁺], suggesting that it inhibited GA action downstream of the Ca²⁺ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca²⁺ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 μm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca²⁺-dependent regulator of the GA response in these cells.     

Gibberellic Acid and Ion Release from Barley Aleurone Tissue: Evidence for Hormone-dependent Ion Transport Capacity

The release of potassium, magnesium, and phosphate ions from aleurone cells of barley (Hordeum vulgare L. cv. Himalaya) is a gibberellic acid-dependent process. The release of these ions is preceded by a lag period of 6 to 8 hours after gibberellic acid addition. The effect of gibberellic acid on the release of ions is not mediated through an effect on ion solubilization. Thus, gibberellic acid does not apreciably affect the sum of extracted and released ions relative to controls. Rather, the effect of the hormone is on the release process itself. Inhibitors of oxidative phosphorylation when added with gibberellic acid or at times up to 6 hours after gibberellic acid inhibition release. When these inhibitors are added after ion release has begun, however, rapid efflux of ions occurs. These results suggest a strong correlation between energy levels and ion transport capacity. Inhibitors of RNA and protein synthesis also inhibit gibberellic acid-stimulated ion release. Evidence suggests that RNA and protein synthesis are required to establish and maintain ion release capacity of aleurone cells.     

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

The gibberellic acid (GA₃)-induced α-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 (α-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 (α-amylases 1 and 2) and group B (α-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 GA₃ 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 GA₃ differentially controls the expression of two α-amylase genes or groups of genes giving rise to two groups of α-amylases with many different properties.

     

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.     

PEP4 Gene Function Is Required for Expression of Several Vacuolar Hydrolases in SACCHAROMYCES CEREVISIAE

The pep4-3 mutation results in a 90–95% reduction in the levels of five vacuolar hydrolases in yeast, including proteinases A and B, carboxypeptidase Y, RNase(s) and the repressible alkaline phosphatase. The mutation is without effect on two secreted glycoproteins, on an enzyme of the vacuolar membrane, and on a proteinase located outside of the vacuole. Mutations at the PEP4 locus exhibit a dosage effect on the levels of some, but not all, of the enzymes whose expression requires the function of the gene.     

牛磺胆酸作用下副溶血弧菌全基因组比较转录谱的表达分析

目的利用DNA芯片技术研究副溶血弧菌对牛磺胆酸刺激反应的全局性基因转录变化概况,找出其中的表达调控变化规律,为副溶血弧菌基因转录调控网络的构建提供实验和理论依据。方法副溶血弧菌分别在正常和添加了50mmol／L牛磺胆酸的培养基中孵育至对数中期,收集菌体,提取RNA,利用全基因组DNA芯片分析比较两者基因转录变化。并应用聚类分析比较其中的变化规律。结果比较转录谱分析证实一共有255个基因的转录表达发生显著性变化,和对照组相比,上调的基因明显占主导优势。而在这些变化的基因中,关于蛋白合成和硫代谢以及谷氨酸合成相关的基因均呈现明显的转录上调变化。结论我们利用DNA芯片技术描绘出了副溶血弧菌在添加牛磺胆酸后全部基因转录水平变化的概图,并发现了蛋白合成,硫代谢和谷氨酸合成相关的基因的变化规律,这给我们下一步的转录调控网络研究提供了良好的靶标。     

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.     

Influence of Gibberellic Acid and the Rht3 Gene on Choline and Phospholipid Metabolism in Wheat Aleurone Tissue

The effect of gibberellic acid (GA3) on phospholipid metabolismand -amylase production was studied in aleurone tissue of twonear-isogenic lines of wheat (Triticum aesuvum L.). Incubationof embryoectomized seeds from a GA-responsive line (rht3, tall)with GA₃ caused the induction of -amylase activity after a lagphase of 30 h. In the case of embryoectomized seeds from a ‘GA-insensitive’line (Rh13, dwarf), however, the lag phase was extended up to50 h. During the first 14 h following imbibition, GA₃ inhibitedcholine uptake and its subsequent incorporation into phosphatidylcholine in the Rhr3 line but not in the rht3 line. GA₃ promotedphospholipid breakdown in both the lines during this period,however. GA₃ also terminated independent turnover of the cholineN-methyl groups in phosphatidyl choline and promoted turnoverof the whole choline headgroup. These results are discussedin relation to the possibility that phosphatidyl choline turnoveris an integral part of the GA₃ signal-transduction mechanismin aleurone tissue. Key words: GA3, Rht3 gene, choline, phospholipid     

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.     

alpha-Amylase Isozymes in Gibberellic Acid-treated Barley Half-seeds

The presence of multiple forms of α-amylase in gibberellic acid-treated embryoless barley half-seeds was demonstrated by separation on diethylaminoethyl-Sephadex and isoelectric focusing polyacrylamide gel disc electrophoresis. Two major α-amylase fractions (A and B), each consisting of two to three isozyme components, were purified. α-Amylase fractions A and B were distinguishable in their reaction patterns. The optimal pH of fraction A α-amylase was found to reside in the acidic side (pH 5.0), as was determined by analyzing the reducing sugars formed as well as the paper chromatographic detection of reaction products. At neutral pH, 6.9, fraction A exhibited weak amylolytic activity in forming maltose. The α-amylase activity in fraction A was markedly stimulated by heat treatment (70 C/15 minutes). Fraction B, constituting a major part of amylases in the endosperm extract, was also found to be composed of α-amylase, as evidenced by the loss of enzyme activity upon allowing fractions A and B to stand at pH 3.3 for a prolonged period. The possible physiological function of the two different types of α-amylase in the carbohydrate breakdown of barley seeds is discussed.     

MyD88 Signaling in the CNS Is Required for Development of Fatty Acid-Induced Leptin Resistance and Diet-Induced Obesity

Obesity-associated activation of inflammatory pathways represents a key step in the development of insulin resistance in peripheral organs, partially via activation of TLR4 signaling by fatty acids. Here, we demonstrate that palmitate acting in the central nervous system (CNS) inhibits leptin-induced anorexia and Stat3 activation. To determine the functional significance of TLR signaling in the CNS in the development of leptin resistance and diet-induced obesity in vivo, we have characterized mice deficient for the TLR adaptor molecule MyD88 in the CNS (MyD88^ΔCNS). Compared to control mice, MyD88^ΔCNS mice are protected from high-fat diet (HFD)-induced weight gain, from the development of HFD-induced leptin resistance, and from the induction of leptin resistance by acute central application of palmitate. Moreover, CNS-restricted MyD88 deletion protects from HFD- and icv palmitate-induced impairment of peripheral glucose metabolism. Thus, we define neuronal MyD88-dependent signaling as a key regulator of diet-induced leptin and insulin resistance in vivo.