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.     

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.     

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.     

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.     

NPA Inhibits Secretion of Amylases by Barley Aleurone Cells and Auxins can Overcome this Inhibition

Polar cell growth is regulated by auxin (Klämbt et al., 1992). It is essentially dependent upon exocytosis. Is exocytosis directly regulated by auxin? Aleurone cells secrete amylases, as well as other hydrolases, by exocytosis. In preliminary experiments, with 22 h of incubation, increasing concentration of N-1-naphthylphthalamic acid (NPA) parallels the inhibition of amylase secretion. A model system for secretion was designed. Barley aleurone layers, previously incubated in 1 μM GA₃ for 16 h, then washed extensively in the presence of 10 μM NPA for 10 min, were incubated finally for 2 h in GA₃, NPA and/or auxins. NPA inhibits amylase secretion. Auxins completely abolished secretion inhibition at the optimal concentration of 1 μM. Aleurone layers contain 1.5 to 3 μM endogenous indole-3-acetic acid (IAA) and have a high capacity to accumulate and retain IAA. A 7-fold accumulation occurred within 3 h after application of 20 μM radiolabeled IAA. A loss of about 10% could be measured within the following 4 h period in fresh media, independent of the presence of NPA. The inhibition of amylase secretion by NPA is not due to an inhibition of protein biosynthesis which was tested by the use of [³⁵S]-methionine.     

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.     

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 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.     

The Structure of Plant Cell Walls: VII. Barley Aleurone Cells

The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls.     

The Induction of Glyoxysomal Enzyme Activities in the Aleurone Cells of Germinating Wheat

The aleurone cells of quiescent Triticum vulgare grain wereobserved to contain glyoxysomes, but enzymes known to be locatedin this organelle were not detected. During germination thenumber of glyoxysomes increased, and their associated enzymeactivities appeared, increasing up to the fifth or sixth day.The appearance of ß-oxidation, isocitratase, and malatesynthetase activities were largely dependent upon the presenceof the embryo. Gibberellic acid (GA₂) was effective in replacingthe embryo in this role. It is proposed, therefore, that thedevelopment of glyoxysomal enzyme activities and probably ofthe glyoxysomes themselves, is a gibberellic acid-dependentprocess. The developments of citrate synthetase and malate dehydrogenaseactivities were only partly dependent upon gibberellic acid.Since it is known that these enzymes are located in other compartmentsbesides the glyoxysomes, it is proposed that their gibberellicacid-dependent activities are located in glyoxysomes while theirgibberellic acidindependent activities are located in the cytosoland/or the mitochondria. The developmental courses of the gibberellicacid-independent activities and the results of studies usinginhibitors of protein synthesis support this hypothesis     

Novel Phosphoinositides in Barley Aleurone Cells (Additional Evidence for the Presence of Phosphatidyl-scyllo-Inositol)

A novel isomer of phosphatidylinositol that differs in the structure of the head group was detected in barley (Hordeum vulgare cv Himalaya) seeds. In this paper we describe our efforts to elucidate the structure of the novel isomer. Evidence from a variety of techniques, including chemical modification of in vivo 32Pi- and myo-[3H]inositol-labeled compounds, gas chromatography-mass spectrometry analysis, in vivo incorporation of scyllo-[3H]inositol, and enzymatic studies that suggest that the structure is phosphatidylscyllo-inositol (scyllo-PI), is presented. The use of microwave energy to significantly enhance the slow rate of hydrolysis of phosphoinositides is described. The presence of scyllo-PI can be easily overlooked by the methods commonly employed; therefore, experimental considerations important for the detection of scyllo-PI are discussed.     

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.     

NaCl-and Gibberellic Acid-induced Changes in the Content of Auxin and the Activities of Cellulase and Pectin Lyase During Leaf Growth in Rice (Oryza sativa)

The interactive effect of NaCl salinity and gibberellic acidin the activities of cellulase and pectin lyase, and on thecontent of auxin and chlorophyll, has been determined duringleaf growth (fifth from base) in rice. The linear growth, chlorophyllcontent, activity of cellulase, and the auxin level of leaveswere markedly decreased when plants were exposed to salt stress(12 dS m^–1). However pectin lyase activity did not registerany significant alteration in the leaves of salt-stressed plantscompared with the control. Treatment of plants with gibberellicacid (GA₃) (10 ppm) increased the leaf growth and chlorophyllcontent with a concomitant rise in the activity of cellulaseunder stressed as well as non-stressed conditions. A markedincrease in the content of auxin was discernible in the leavesof salt-stressed plants treated with GA₃ compared with non-treatedsalinized ones. An appreciable increment in the activity ofpectin lyase in response to GA₃ administration was detectedonly in the leaves of non-stressed plants. These results indicatethat enhancement of cellulase activity and the augmentationof endogenous auxin content may be involved in the stimulationof rice leaf growth by GA₃ under saline conditions. Oryza sativa, rice, leaf growth, NaCl salinity, gibberellic acid, cellulase, pectin lyase, auxin     

Modulation of Calmodulin mRNA and Protein Levels in Barley Aleurone

Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone protoplasts. Monoclonal antibodies to tubulin and polyclonal antibodies to tonoplast intrinsic protein and malate synthase were used as controls. CaM was localized to the nucleus, the vacuolar membrane, and the cytosol, but was not associated with microtubules.     

Abnormalities Induced in Barley Ears by Gibberellic Acid

Five varieties of spring barley were sprayed with gibberellicacid (GA) at various growth stages from the one-leaf to thefive-leaf stage. In short days a number of abnormalities ofthe ear were found. These abnormalities are described in themature ear, and in the case of the branched ear, the ontogenyof the abnormality is shown. The relationship of the abnormalitiesto the environment, to the stage at which the plant was treated,and to the variety is analysed. The role of CA in inducing these abnormal features is discussedand their resemblance to abnormalities induceed geneticallyand by enviromental manipulations is pointed out.     

Changes of Plastids During Xylem Differentiation in Barley Root

Plastids (eoplasts) are present in meristematic cells of prospectivecentral metaxylem in the barley root. Starch starts to be formedin plastids precisely after the cessation of mitotic activityand at the beginning of endomitotic growth. During secondarywall formation, the starch is gradually lost. Cavities are formedin plastids and signs of plastid degeneration are present fromthis stage of cell development. However, some intact globularplastids without starch are present until shortly before thefinal step of ontogeny, i.e. total destruction of protoplast. Hordeum distichum L., root, xylem, plastids, endomitotic growth, starch     

Characterization of the Major Protein Component from Aleurone Cells of Barley (Hordeum vulgare L.)

Comparison of the SDS-PAGE patterns of salt-soluble proteinsfrom aleurone protoplasts, enriched aleurone layers preparedby pearling, and isolated embryos of mature barley showed threegroups of bands that reacted with antiserum raised against the7S globulins of oat embryos. These had M_rs of about 50 000,40 000 and 25 000. The enriched aleurones also contained a thirdgroup of immunoreactive bands (M_r about 70 000), which did notco-purify when the proteins were fractionated by ammonium sulphateprecipitation, ion exchange chromatography and immunoaffinitychromatography. The purified protein gave a single sharp peakon RP-HPLC, and contained a fourth minor group of subunits ofM_r about 20 000, in addition to those of M_rs about 50 000, 40000 and 25 000. The holoprotein and the ‘major groups’ of subunitsall had similar major N-terminal amino acid sequences whichwere related to the N-terminal amino acid sequences of pea andbean vicilins, and to sequences in the putative N-terminal regionsof the mature 7S ¹-globulins of cottonseed, confirming the homologywith these groups of proteins. Key words: Barley, seeds, 7S globulin, vicilin, ¹-globulin, amino acid sequence, protein homology     

Phosphoinositides in Barley (Hordeum vulgare L.) Aleurone Tissue

[3H]Inositol labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers and analysis of phospholipids by deacylation revealed the presence of phosphatidylinositol (PtdIns), PtdIns3P, and PtdIns4P but not PtdInsP2 species. In contrast to an earlier report (P.P.N. Murthy, G. Pliska-Matyshak, L.M. Keranen, P. Lam, H.H. Mueller, N. Bhuvarahamurthy [1992] Plant Physiol 98: 1498-1501) systematic chemical degradation of PtdIns revealed no evidence of a second isomer of PtdIns. Evidence of the widespread occurrence of 3-phosphorylated PtdIns within the plant kingdom is presented.