Oriented structure of pectic polysaccharides in pea epidermal cell walls

Polarized infrared absorption spectra of film specimens of theepidermal cell wall of the third internode of pea stems wererecorded before and after treatment with endopolygalacturonase(endo-PG) and endo-pectin lyase (endo-PL). The spectra showedthat the pectic polysaccharides solubilized with endo-PG wereessentially the same as those solubilized with endo-PL. Thedegree of esterification of the pectic polysaccharides was about20%, and their major sugar components were uronic acids (32.8%),arabinose (48.1%) and galactose (19.2%). The polarized infraredspectra showed that pectic polysaccharides have an orientedstructure in cell walls with their molecular chains orientedpreferentially parallel to the direction of cell elongation. ¹Present address: Research and Development, Kanzaki Paper Mfg.Co., Ltd., Amagasaki, Hyogo 660, Japan. ²Present address: Wakayama Research Laboratories, Kao Soap Co.,Ltd., Wakayama 640-91, Japan. (Received June 28, 1980; )     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Transfer RNA, a Possible Supplier of Free Cytokinins, Ribosyl-cis-zeatin and Ribosyl-2-methylthiozeatin: Quantitative Comparison between Free and Transfer Cytokinins in Various Tissues of the Hop Plant

Cytokinins in both free pool and tRNAs have been identifiedand quantified in the unfertilized cone, fertilized cone, seed,cotyledon, epicotyl and root of the hop plant (Humulus lupulusL. cv. Shinshu-wase) on the basis of combined gas chromatography-massspectrometry and combined gas chromatography-selected ion currentmonitoring analysis. Based on the quantitative comparison betweenfree pool cytokinins and tRNA cytokinins, it is suggested thatribosyl-cis-zeatin and ribosyl-2-methylthiozeatin might be derivedfrom tRNA catabolism and that the level of ribosyl-trans-zeatinmight be controled independently of the tRNA catabolism. ¹ Present address: Taisho Pharmaceutical Co. Ltd., Yoshini-cho,Omiya-shi, Saitama 330, Japan. (Received December 1, 1981; Accepted February 9, 1982)     

Properties and Possible Physiological Significance of Cell Wall Calcium Binding in Etiolated Pea Epicotyls

Baydoun, E. A-H. and Brett, C. T. 1988. Properties and possiblephysiological significance of cell wall calcium binding in etiolatedpea epicotyls.—J. exp. Bot. 39: 199–208. The binding of ⁴⁵Ca²⁺ ions to cell walls prepared from pea epicotylswas examined in young and old parts of the epicotyl, and wasfound to be considerably greater, on a carbohydrate basis, inthe older, non-growing cells. A similar comparison between light-and dark-grown stems showed greater binding in the dark-grownstems. The polygalacturonase-insensitive component of the bindingcontained at least three types of binding with different affinities,and had an apparent pK of 4.3. The specificity of the bindingfor calcium ions was examined and a considerable degree of specificitywas observed. The specificity of inhibition by calcium of epicotylelongation was similar to the specificity of calcium binding.A specific calcium chelator, EGTA, when present at a concentrationof above 10 mol m^–3, promoted the extension of matureregions of the epicotyl, while inhibiting extension of youngertissue. Key words: Cell wall, calcium, pea epicotyl     

Combined Effects of Multiple cis-Acting Elements in Elicitor-Mediated Activation of PSCHS1 Gene

Promoter of a gene encoding chalcone synthase 1 (PSCHS1), amember of the defense-related genes in pea, was analyzed bytransient transfection assay. The results demonstrated thatin addition to the previously identified AT-rich sequences,at least four distinct cis-acting elements were required formaximal fungal elicitor-mediated activation of PSCHS1. ²Present address: Hikone Reserch Laboratories, Maruho Co. Ltd.,2763, Takamiya-cho, Hikone, Shiga, 522-02 Japan.     

Differential Modulation of Carboxylase and Oxygenase Activities of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Released from Freshly Ruptured Spinach Chloroplasts

The carboxylase activity of ribulose 1,5-bisphosphate (RuBP)carboxylase/oxygenase released from freshly ruptured spinachchloroplasts was stimulated preferentially by Mg²⁺ while oxygenaseactivity was higher with Mn²⁺. Only Mg²⁺ could reactivate eitheractivity of desalted enzyme. The results suggest that carboxylaseand oxygenase activities of RuBP craboxylase/oxygenase can bemodulated selectively by Mg²⁺ or Mn²⁺. ¹ Present address: Department of Botany, Sri Venkateswara University,Tirupati 517 502, India. (Received March 5, 1981; Accepted June 26, 1981)     

The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)     

Partial purification and some properties of an L-{alpha}-amino-S-caprolactam-hydrolyzing enzyme from Cryptococcus laurentii

A new L-amlnolactam-hydrolyzing enzyme was partially purifiedfrom cells of Cryptococcus laurentii which can grow on L-aminolactamas a carbon and nitrogen source. The enzyme required a bivalentmetal ion, such as Mn²⁺ or Mg²⁺, and its molecular weight wasroughly estimated to be 1.5?10⁵ Some other properties were alsostudied. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University Sumiyoshi-ku, Osaka 558, Japan. (Received June 20, 1977; )     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

Phosphoenolpyruvate Carboxylase of Maize Leaves: an Improved Method for Purification and Reduction of the Inhibitory Effect of Malate by Ethylene Glycol and Bicarbonate

Light-adapted and dark-adapted forms of phosphoenolpyruvatecarboxylase were purified from maize leaves by an improved methodthat included chromatography on Butyl-Toyopearl in the presenceof ethylene glycol. The inhibition by malate was relieved notonly by increasing concentrations of ethylene glycol but alsoby bicarbonate at pH 7.0. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-32 Japan. ²Present address: Asahi Medical Co., Ltd., 4-3400-I Asahimachi,Nobeoka, 882 Japan. ³Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412 Japan.     

Salicylic Acid Induces Extracellular Superoxide Generation Followed by an Increase in Cytosolic Calcium Ion in Tobacco Suspension Culture: The Earliest Events in Salicylic Acid Signal Transduction

Addition of salicylic acid (SA) to tobacco (Nicotiana tabacum)suspension culture immediately induced a rapid and transientgeneration of superoxide anion (O^–₂), followed by a transientincrease in cytosolic free calcium ion concentration ([Ca²⁺]_c).The level of SA-induced O^–₂ was lowered by treatment withseveral scavengers of active oxygen species and a peroxidaseinhibitor, but not with an NADPH oxidase inhibitor. The SA-induced[Ca²⁺]_c elevation was also lowered by inhibitors which effectivelylowered the O^–₂ level. Inhibition of [Ca²⁺]_c elevationby Ca²⁺ channel blockers and a Ca²⁺ chelator indicated thatextracellular Ca²⁺ was responsible for the increased [Ca²⁺]_c.Among the several SA analogs, only compounds that actively inducedthe O^–₂ generation also elevated [Ca²⁺]_cIn addition, theinhibitory effects of SA analogs on catalase activity correlatedwell with their effects on the O^–₂ generation and the[Ca²⁺]_c elevation. SA-dependent O^–₂ generation was shownto occur extracellularly, requiring both H₂O₂ and at least oneproteinaceous factor excreted from the cells. This factor wasdetermined to be a salicylhydroxamic acid-sensitive extracellularguaiacol-utilizing peroxidase. ⁴Present address: Isehara Research Laboratory, Kanto ChemicalCo., Inc., Suzukawa, Isehara, 259-1146 Japan.     

Inhibition of Adventitious Root Growth in Tradescantia by Calmodulin Antagonists and Calcium Inhibitors

When cuttings of Tradescantia fluminensis stem were incubatedin distilled water, the buds located at the node grew into adventitiousroots. The root growth could be inhibited by calmodulin antagonists,trifluoperazine, chlorpromazine, compound 48/80 and calmidazolium,in a concentration-dependent manner. The divalent cation chelatorethyleneglycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetraaceticacid had no effect, however, the intracellular chelator TMB-8completely inhibited root growth. The growth was also inhibitedby calcium ionophore A23187 [GenBank] , lanthanum, a competitive inhibitorof Ca²⁺ uptake and verapamil, a calcium channel inhibitor. AWestern blot of the adventitious root extract followed by immunostainingwith an anti-spinach calmodulin antibody clearly showed thepresence of calmodulin in this tissue. These results stronglysuggest the involvement of calmodulin and calcium in the growthof Tradescantia advenitious roots. ¹A part of this work has been published in abstract form in"Molecular and Cellular Aspects of Calcium in Plant Development"(Editedby A.J.Trewavas, Plenum Publishing Co. 1986. ³Present address: Plant Laboratory , Kirin Brewerry Co. Ltd.,Kitsuregawa-cho, Tochigi-ken 329-14 ,Japan (Received May 2, 1987; Accepted September 17, 1987)     

Membrane Potentials in Excitable Cells of Aldrovanda vesiculosa Trap-Lobes

The resting membrane potential in excitable cells of Aldrovandatrap-lobes is composed of diffusion and electrogenic potentials.The diffusion potential, about –100 mV in artificial pondwater, was determined from the external K⁺ and Na⁺ concentrations.The permeability ratio, P_Na/P_K of the membrane was estimatedto be about 0.3. The electrogenic potential hyperpolarized themembrane to about –140 mV. The peak value of the actionpotential increased by +26 mV with a tenfold increase in theexternal Ca²⁺ concentration. The action potential was blockedby an application of the Ca²⁺ chelater or the Ca channel blocker,LaCl₃. Cells showed additional Ca²⁺ influx (7.8 pmole/cm² impulse)during membrane excitation. These facts suggest that the transientincrease in Ca²⁺ influx causes the action potential presentin cells of Aldrovanda trap-lobes. ¹ Present address: Jerry Lewis Neuromuscular Research Center,School of Medicine, University of California Los Angeles, LosAngeles, CA90024, U.S.A. ² Present address: Biological Laboratory, Kyoritsu Women's University,Hachioji 193, Japan. (Received September 21, 1983; Accepted September 7, 1984)     

The Deduced Amino Acid Sequence of Phytochrome from Adiantum Includes Consensus Motifs Present in Phytochrome B from Seed Plants

A cDNA for the phytochrome of the fern Adiantum capillus-venerisL. was cloned and sequenced. The deduced phytochrome is 50{smalltilde}55% identical to phytochromes of seed plants, and 68%identical to Selaginella phytochrome. Regions resemble thosein previously characterized phytochromes from ferns, lower plantsand seed plants. ³Present address: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,Tsukuba-shi, Ibaraki, 305 Japan ⁴Present address: Plant Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,Saitama, 351-01 Japan ⁵Present address: Advanced Research Laboratory, Hitachi, Ltd.,Hatoyama, Saitama, 350-03 Japan     

Purification and Properties of UDP-glucuronate Pyrophosphorylase from Pollen of Typha latifolia Linne

UDP-glucuronate pyrophosphorylase from the pollen of Typha latifoliaLinne was purified about 600-fold by protamine sulfate treatment,ammonium sulfate fractionation, gel filtration, chromatofocusing,affinity chromatography, and isoelectric focusing. The purificationwas carried out using buffer containing 20% sucrose which helpedto prevent enzyme inactivation. This enzyme required equimolarlevels of Mg²⁺ to PP_i or UTP for maximum velocity of enzymecatalysis. Results of experiments on product inhibition andthe initial velocity of the enzyme catalysis reaction suggesteda Theorell-Chance mechanism. ¹ Present address: Japan Spectroscopic Co., 2967-5, Ishikawa-Cho,Hachioji-City, Tokyo 192, Japan. (Received April 5, 1983; Accepted September 26, 1983)     

Wounding Inhibits Protein Synthesis yet Stimulates Polysome Formation in Aged, Excised Pea Epicotyls

Wounding of aged, previously-excised pea epicotyl segments byremoval of the basal 1–2 mm resulted in a rapid (beginningwithin 15 min) recruitment of monosomes on to polysomes andan even more rapid (maximal between 6–12 min) inhibitionof protein synthesis in the remaining tissue. This inhibitionof protein synthesis in vivo did not appear to be an artefactcaused by the removal of highly active tissue (e.g., callus,contaminating bacteria), since wounds inflicted at a site distantfrom the region analyzed still elicited the response, and proteinsynthesis in the 1–2 mm slices (normally discarded) wasinhibited even more strongly than it was in the remaining tissue.The proportion of radioactive methionine in nascent chains (boundto polysomes) increased, while the production of completed polypeptidesdecreased, after wounding. Cycloheximide, a known inhibitorof the ribosome translocation/release process mimicked someof the effects of wounding. We interpret the results to indicatethat the initial effect of wounding is to inhibit translationby inhibiting the ribosome translocation/release process, whereasthe subsequent recovery in protein synthesis is brought aboutpartly by a recovery in ribosome translocation/release and partlyby enhanced initiation. ¹ Present address: Harvard-MIT Division of Health Science andTechnology, MIT, Cambridge, Massachusetts 02139, U.S.A. ² Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received May 26, 1986; Accepted August 4, 1986)     

Control of Glutamate Dehydrogenase from Green Tobacco Callus Mitochondria by Ca2$ and pH

Glutamate dehydrogenase (GDH) (EC 1.4.1.3 [EC] .) purified from greentobacco callus mitochondria was activated markedly by Ca^2$ inthe amination reaction. This activation was detectable evenat concentrations below 5 µM Ca^2$. Saturation curves for the three substrates of the aminationreaction showed normal Michaelis-Menten kinetics in the presenceof 1 mM of Ca^2$, but pronounced substrate inhibition occurredwithout Ca^2$. The effect of Ca^2$ was chiefly on the maximalvelocity. The saturation curve for NH₄Cl in the presence of Ca^2$ was modulatedby a change in pH. The apparent K_m value for NH₄Cl markedlydecreased whereas that for -ketoglutarate increased slightlywhen the pH was raised from 7.3 to 9.0. In contrast, the K_mfor NADH was little affected by raising the pH. The characteristicof GDH which increases its affinity for NH₄Cl when the pH israised may be compatible with the detoxification of ammonia. ¹ Present address: Mochida Pharmaceutical Co., Ltd. (Received August 24, 1981; Accepted November 28, 1981)     

Effect of Ca2+ on Tonoplast Potential of Permeabilized Characeae Cells

Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca²⁺ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca²⁺, the tonoplast potential(E_M became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl^–-channelinhibitor, E_m became more negative and the response of E_m toCa²⁺ was significantly suppressed. It is suggested that Ca²⁺activates Cl^–-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl^–-channelin N. axilliformis. ¹Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)