RNA Polymerase Activity During Breakage of Seed Dormancy by Low Temperature Treatment of Fruits of Acer platanoides (Norway Maple)

Slater, R. J. and Bryant, J. A. 1987. RNA polymerase activityduring breakage of seed dormancy by low temperature treatmentof fruits of Acer platanoides (Norway maple).—J. exp.Bot. 38:1026–1032. Endogenous RNA polymerase activity has been characterized innuclei isolated from embryo axes of Acer platanoides. Optimalactivity was recorded at 4·0 mol m^–3 MgCl₂ and50 mol m^–3 (NH₄)₂SO₄ and total activity could be inhibitedby up to 30% by -amanitin. Stratification of fruits leads toa stimulation of RNA polymerase activity. A minimum of 3 d coldtreatment is required with at least 3-fold stimulation recordedafter 10 d at 4°C. The increased enzyme activity is resistantto -amanitin suggesting an effect on RNA polymerase I. Key words: Acer platanoides, RNA polymerase, seed dormancy     

In vivo Studies on the Occurrence of Stored mRNA in Embryonic Axes of Vigna unguiculata Seeds

-Amanitin and cordycepin at various concentrations were testedfor their inhibitory effect on the fresh weight increase ofVigna unguiculata embryonic axes after the onset of imbibitionand on the incorporation rate of ³H-labeled leucine into proteinin axes of the 36–38 h stage. -Amanitin at 0.5–5µ/Kg/ml clearly exerted an inhibitory effect on both thefresh weight increase and the protein synthesis. This drug at1 µg/ml, however, showed no significant effect on theprotein synthesis at an early stage of imbibition (4 h), whereascycloheximide was a very potent inhibitor. By experiments inwhich ‘dry’ axes were allowed to imbibe 3H-lebeledadenosine solution for 4 and 12 h in the presence of -amanitin,it was found that poly A⁺RNA was newly synthesized to some extentin axes as early as 4 h after the onset of imbibition and thatthe drug effectively inhibited the poly A⁺RNA synthesis. Theresults may indicate the occurrence of stored mRNA in embryonicaxes of V. unguiculata seeds. (Received June 11, 1983; Accepted August 16, 1983)     

Diurnal rhythm of nuclear RNA polymerase I activity in a duckweed, Lemna gibba G3, under continuous light conditions

The diurnal change of nuclear RNA polymerases I and II was examinedin a longday duckweed, Lemna gibba G3, under continuous lightconditions. RNA synthesis in crude nuclei was dramatically stimulatedby addition of the exogenous RNA polymerase of Escherichia coli,but not by the addition of calf thymus DNA. Treatment of crudenuclei with the supernatant fractions after precipitation ofthe nuclei decreased the RNA synthetic activity of the nucleiirrespective of the preparation time of both fractions. RNApolymerase I activity in crude nuclei, which was determinedin the presence of -amanitin at a low concentration of KCl,exhibited a diurnal rhythm but RNA polymerase II activity, whichwas presumed to be a portion of RNA synthesis inhibited by -amanitinin the presence of a high concentration of KCl, remained constantthroughout the day. Identical results were obtained when bothenzymes were solubilized widi ammonium sulfate and chromatographedon DEAE-Sephadex column. Both activities in the supernatantfraction obtained after precipitation of the nuclei did notchange diurnally. It was concluded, therefore, that the diurnalrhythm of RNA synthetic activity in the crude nuclei is dueto the RNA polymerase I activity and not the RNA polymeraseII activity. (Received August 29, 1978; )     

Increases in activities of membrane-bound hydrolases in pea cotyledons during germination

Membrane-bound proteinase and acid phosphatase activities, butnot cytosol proteinase activity, in pea cotyledons increasedafter lag phases during germination. The activity hydrolyzingN--benzoyl-D,L-arginine P-nitroanilide in the membrane fractionincreased rapidly in the imbibition stage. Whether the increasesare due to de novo synthesis of the enzyme proteins was studied. ¹ Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received May 28, 1973; )     

Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Characterization of the Inhibitory Effect of Aphidicolin on Transdifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

Inhibition by aphidicolin (APC), an inhibitor specific for -typeDNA polymerase, of trans-differentiation into tracheary elementswas characterized in Zinnia mesophyll cells. APC was effectivewhen given in the first 24 h of culture and exposure continueduntil the 36th hour. This suggests temporal involvement of -typeDNA polymerase in transdifferentiation. ¹Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejiro, Tokyo,112 Japan     

Biosynthetic mechanism of glycolate in Chromatium III. Effects of {alpha}-hydroxy-2-pyridinemethanesulfonate, glycidate and cyanide on glycolate excretion

Effects of -hydroxy-2-pyridinemethanesulfonate (-HPMS), 2,3-epoxypropionate(glycidate), and cyanide on the photosynthetic activities ofChromatiumwere studied. -HPMS stimulated photosynthetic CO₂fixation in the bacterial cells in both N₂ and O₂ environments.The formation and subsequent excretion of both glycolate andglycine in the O₂ atmosphere were markedly enhanced by -HPMS.In contrast to a recent report by Zelitch [Arch. Biochem. Biophys.163: 367–377 (1974) ] that glycidate specifically inhibitsglycolate formation in tobacco leaf disks, we found that ithad no influence on CO₂ fixation by Chromatium in either N₂or O₂ atmosphere, and that the synthesis and extracellular excretionof glycolate were markedly stimulated by glycidate treatment.Cyanide (0.01–1 mM) exerted a marked inhibitory effecton photosynthetic CO₂ fixation in N². In O₂ atmosphere, photosynthesiswas stimulated by 0.01 mM cyanide, and inhibited by it abovethis level. Both the incorporation of ¹⁴CO₂ into glycolate andthe total synthesis of glycolate in the light were also enhancedby 0.01 mM cyanide, and strongly inhibited above that concentration. ¹This is paper XXXVI in the series "Structure and Function ofChloroplast Proteins," and the research supported in part bygrants from the Ministry of Education of Japan (No. 111912),the Toray Science Foundation (Tokyo) and the Naito Science Foundation(Tokyo). (Received May 31, 1976; )     

Metabolic Changes in Axes of Germinating Vigna unguiculata Seeds as Related to Effects of Removal of Cotyledons

[¹⁴C]-Labeled amino acids and sucrose were fed to Vigna unguiculataseeds through cut-ends of cotyledons, and incorporations ofradioactivity into trichloroacetic acid- and 80% ethanol-insolublefractions of axes, respectively, were followed during 48 h ofthe post-imbibition development. The results of these studies,together with determinations of changes in dry weight and proteincontents after the onset of imbibition, indicated that the reservematerials stored in cotyledons were available for active growthof axes only after 12 h of post-imbibition. However, pulse-labelingexperiments, where [³H]-labeled leucine and uridine were feddirectly to axes attached to or detached from cotyledons, indicatedthat synthesis of protein and RNA in both axes was very pronouncedeven at earlier stages (2–8 h) of post-imbibition. Albuminand globulin proteins of axes disappeared most rapidly duringthe 6–12 h period of post-imbibition. Cycloheximide, -amanitinand cordycepin added to imbibing axes inhibited the degradationof major globulin proteins, whereas the inhibitors had littleeffect on the degradation of major albumin proteins. Both proteolyticand amylolytic activities were found to occur in embryonic axesof ‘dry’ seeds, and increased to higher levels asthe germination proceeded. Axes at early stages of germinationmay degrade the self-sustained reserve proteins and utilizethem for the synthesis of new proteins. (Received June 11, 1983; Accepted August 16, 1983)     

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)     

Permeant but not impermeant divalent cations enhance activation of nondesensitizing alpha(7) nicotinic receptors

Neuronal₇ nicotinic acetylcholine receptors (nAChRs) arepermeable to Ca²⁺ and other divalent cations. Wecharacterized the modulation of the pharmacological properties ofnondesensitizing mutant (L²⁴⁷T andS²⁴⁰T/L²⁴⁷T) ₇ nAChRs bypermeant (Ca²⁺, Ba²⁺, and Sr²⁺) andimpermeant (Cd²⁺ and Zn²⁺) divalent cations.₇ receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,whereas impermeant divalent cations decreased potency and maximalefficacy. The antagonist dihydro--erythroidine (DHE) was a strongpartial agonist of L²⁴⁷T andS²⁴⁰T/L²⁴⁷T ₇ receptors in thepresence of divalent cations but was a weak partial agonist in thepresence of impermeant divalent cations. Mutation of the"intermediate ring" glutamates (E²³⁷A) inL²⁴⁷T ₇ nAChRs eliminated Ca²⁺conductance but did not alter the Ca²⁺-dependent increasein ACh potency, suggesting that site(s) required for modulation are onthe extracellular side of the intermediate ring. The difference betweenpermeant and impermeant divalent cations suggests that sites within thepore are important for modulation by divalent cations.

     

RNA Synthesis in Phaseolus Chloroplasts: I. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLAST PREPARATIONS FROM PHASEOLUS VULGARIS L. LEAVES AND SOLUBILIZATION OF THE RNA POLYMERASE

Chloroplast preparations from the young primary leaves of Phaseolusvulgaris L. cv. Canadian Wonder carry out the DNA-dependentincorporation of UTP into RNA at rates between 8 and 14 pmolUTP µg^–1 chlorophyll h^–1. It is estimatedthat 90% of the activity was localized in the chloroplasts.The incorporation proceeded for between 20 and 30 min at 35°C. The maximum rates of RNA synthesis were attained atpH 8.3, in the presence of 15 mM MgCl₂. Chloroplasts were alsoactive, to a lesser extent, with 1.5 mM MnCl₂. The simultaneouspresence of MnCl₂ and MgCl₂ resulted in inhibition of activity.Nuclear material prepared from young P. vulgaris leaves incorporatedUTP at a rate of about 12 pmol UTP µg^–1 DNA h^–1.On a chloroplast (Tritonsoluble) DNA basis chloroplast activitywas over 40-fold that of nuclei. Methods of solubilizing chloroplastRNA polymerase were explored. Yields of over 75% were achieved,but methods suitable for one species were not always successfulwhen applied to another. The highest yields of the P. vulgarisenzyme were obtained using EDTA and KCl. All methods resultedin solubilization of DNA. RNA synthesis by the soluble P. vulgarisenzyme proceeded for more than 40 min at 35 °C.     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

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)     

Growth Inhibition by Lysine Plus Threonine: Comparative Inhibition Profiles between Floury-a and Normal Maize Embryos

Lysine and threonine, either alone or in combination, were testedfor their effects on growth of floury- mutant and DY normalembryos of maize in culture. At 1 mM of lysine, no inhibitoryeffect was observed on either type of embryo, although at 2.5mM slight inhibitions were observed, principally on the rootand shoot lengths. The inhibition profiles by threonine from0.5 to 2.5 mM were similar to those by lysine only in the floury-embryos; in the normal DY embryos, threonine produced a markedinhibition, especially on the root and shoot length. In conjunctinhibition experiments, the profile of inhibition obtained forfloury- embryos by variable levels of lysine with a constantlevel of threonine were always identical to that by variablelevels of threonine with a constant lysine level. On the otherhand, in the normal DY embryos, the extent of the inhibitoryeffect by variable levels of lysine with a Constant threoninelevel was much greater than that by variable levels of threoninewith a constant lysine level. The involvement of homoserinedehydrogenase of the floury- embryo in the lesser sensitivityfor feedback inhibition by threonine is discussed. ¹ Paper No. 112 of the Instituto Fitot?cnico de Santa Catalina,Facultad de Agronomia, Universidad Nacional de La Plata. (Received July 7, 1981; Accepted December 21, 1981)     

ORIGIN OF AMINO ACIDS CONSTITUTING CELLULAR PROTEIN IN GERMINATING CONIDIOSPORES OF ASPERGILLUS NIGER

Formation of pool amino acids in germinating spores of Aspergillusniger strain 1617 was investigated. The pool amino acids comprisedmainly glutamic acid and alanine. Small amounts of pyruvateand -ketoglutarate were found to increase almost in parallelwith the course of increase in the amount of free amino acidsup to the stage of onset of active protein synthesis. Asparticglutamictransaminase activity was exhibited even in dormant spores andit developed in response to the increase in cellular protein.Alanine-glutamic transaminase activity, on the other hand, waslacking in dormant spores and appeared at the stage of accumulationof amino acids preceding protein synthesis. It was revealed from the experiments with ³⁵S-labeled sporesthat the dormant spores of this fungus contain two unidentifiedsulfur substances, and the sulfur of these substances is incorporatedinto the sulfur amino acids of the protein synthesized in germinatingspores. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Received September 11, 1959; )     

Natural Ageing: Poly(A) Polymerase in Germinating Embryos of Triticum durum Wheat

The viability of seeds is associated with ageing and storageconditions. A loss of viability is accompanied by slow germination,reduced growth, and a decline in protein and poly(A)⁺RNA synthesis.This paper reports on the activity of poly(A) polymerase indry and germinating embryos of Triticum durum Desf. cv. Cappellicaryopses of different ages and viability. The enzyme was presentas a single form during ageing and germination. The poly(A)polymerase was active at decreasing levels in all aged dry embryos,in parallel with loss of viability. Its activity strongly increasedduring the germination only in viable embryos. The observedincrease was due to de novo synthesis of the enzyme. Poly(A)polymerase synthesis was low during germination of less viableembryos and absent in older ones. Reduced poly(A) polymeraseactivity in dry or germinated wheat embryos may cause a shorteningof poly(A) chains in vitro and a decline in poly(A)⁺RNA synthesis.Copyright1995, 1999 Academic Press Triticum durum Desf. cv. Cappelli, wheat, embryo, natural ageing, poly(A) polymerase