Polyamines and Anaerobic Elongation of Rice Coleoptile

The role of polyamines in the anaerobic elongation of rice (Oryzasativa L.) coleoptiles was studied. The reduced growth of ricecoleoptiles under anoxic conditions was accompanied by a massiveaccumulation of free putrescine. Putrescine was synthesizedfrom arginine in a reaction catalyzed by arginine decarboxylase(ADC). The anoxic titer of putrescine was closely correlatedwith elongation of coleoptiles. In experiments in which putrescineand inhibitors [-difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO)] of the synthesis of polyamines were exogenously supplied,we demonstrated an absolute requirement for putrescine, synthesizedby ADC, for anaerobic elongation of coleoptiles. The presenceof exogenous putrescine (alone or in combination with DFMA)increased the rate of anaerobic elongation of coleoptile by30–40%. (Received December 1, 1988; Accepted June 19, 1989)     

Inhibition of Uredospore Germination and Germ Tube Growth by Inhibitors of Polyamine Metabolism in Uromyces phaseoli L

We have studied the effects of two polyamine biosynthetic inhibitors,-difluoromethylor-nithine (DFMO) and -difluoromethylarginine(DFMA), and of polyamines (PAs), alone and in combination onuredospore germination and germ tube growth in Uromyces phaseoliL, race O. Both the inhibitors at concentrations 0.01, 0.1 and1.0 mM produce successively inhibition of uredospore germinationin vitro. The inhibitors also delay the timing of spore germinationfor 15–30 min and restrict germ tube elongation. Stimulationof spore germination and germ tube growth was noticed in culturescontaining PAs (putrescine or spermidine) alone, while culturesfortified with inhibitor plus PA resulted in a partial reversionof the inhibitory effect, suggesting that PAs may be requiredfor normal germination and outgrowth of fungal spores. Sporegermination was completely inhibited on the surface of unifoliolatebean leaves treated with 0.5 mM or higher DFMO 1 d before inoculationwith pathogen, while DFMO treated 1 d after inoculation showedgreater damage of uredosporelings. In contrast, DFMA confersno effect even at 5 mM. Spores collected from bean plants givena pre- and post-inoculatory treatments with DFMO and DFMA showno significant differences in germination and pathogenicity,however, the higher doses caused significant decrease. (Received April 25, 1988; Accepted October 20, 1988)     

Polyamine Biosynthetic Enzymes and the Effect of their Inhibition on the Growth of Some Phytopathogenic Fungi

Studies were conducted on the distribution of two polyaminebiosynthetic enzymes, or-nithine decarboxylase (ODC) and argininedecarboxylase (ADC), and the effect of their inhibitors on growthand polyamine biosynthesis in four phytopathogenic fungi, namely,Helminthosporium maydis, H. carbonum, Fusarium oxysporum f.sp. lycopersici and Ceratocystis ulmi. Three species had highlevel of ODC as compared to ADC activity; in C. ulmi on theother hand, ADC was predominant with very little or no ODC activity.DL--difluoromethylornithine (DFMO) significantly inhibited ODCactivity in all species in vitro with little effect on ADC activity.ADC in all cases was inhibited by DL--difluoromethylarginine(DFMA) but not by DFMO. Mycelial growth of all fungi was inhibitedby 1 to 5 mM concentrations of either DFMO or DFMA within twodays except in H. maydis which remained unaffected even by thehighest concentration (5 mM) of DFMA. In general, the inhibitionwas more pronounced with DFMO as compared to DFMA. Putrescinecompletely reversed the inhibitory effects of DFMO and DFMAin all species. Among the polyamines, spermidine was predominantin all fungi. The cellular concentrations of putrescine andspermidine were considerably lower in the presence of eitherof the inhibitors while spermine levels were higher than thecontrol. ¹Scientific contribution number 1529 from the New HampshireAgricultural Experiment Station. (Received November 25, 1988; Accepted April 11, 1989)     

Polyamine Biosynthesis during Vegetative and Floral Bud Differentiation in Thin Layer Tobacco Tissue Cultures

Levels of putrescine, spermidine and spermine increase whenvegetative or floral buds form in cultures derived from surfaceexplants of inflorescences of Nicotiana tabacum L. cv. Wisconsin-38.Concomitantly, the activity of arginine decarboxylase (ADC)rises and that of ornithine decarboxylase (ODC) declines. DL--Difluoromethylarginine(DFMA), a specific suicide inhibitor of ADC, inhibits bud initiation,while DL--difluoromethylornithine (DFMO), the analogous suicideinhibitor of ODC, does not. On the other hand, DFMO inhibitsthe subsequent development of newly regenerated floral buds,while DFMA does not. It thus appears that polyamines derivedthrough ADC may be involved in bud initiation, while polyaminesderived through ODC are required for subsequent growth and developmentof such buds. Especially large increases of spermidine are associatedwith floral bud differentiation, indicating a possible specialmorphogenetic role for that polyamine. ¹Present address: Laboratori de Fisiologia Vegetal, Facultadde Farmacia, Universitat de Barcelona, 08028 Barcelona, Spain (Received April 25, 1988; Accepted August 12, 1988)     

Effects of Metal Chelators on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after being detachedand kept in the dark was studied in terms of the loss of chlorophyll.Chlorophyll was more rapidly degraded in maize than hydrangeaduring the incubation period in the dark. The loss of chlorophyllin the dark was effectively inhibited in both plants by ,'-dipyridyland o-phenanthroline at concentrations between 0.01 and 0.1mM. Three other chelators of iron produced lesser inhibitionand only at higher concentrations. EDTA prevented the loss ofchlorophyll in maize leaves at concentrations above 10 mM, butdid not do so in hydrangea leaves. Detached leaves floated on EDTA, ,'-dipyridyl or o-phenanthrolinesolutions and exposed to light exhibited a marked bleaching.The bleaching was partially inhibited by applying ascorbic acid. (Received December 26, 1981; Accepted October 18, 1982)     

Temperature-Dependent Inhibitive Actions of {alpha},{alpha}'-Dipyridyl and Cycloheximide on the Senescence of Maize Leaves

Temperature-dependent inhibitive actions of ,'-dipyridyl andcycloheximide on the senescence of maize leaves were studied.,'-Dipyridyl effectively inhibited the loss of chlorophyll at25?C but not at 35?C. Gycloheximide was highly effective inpreserving chlorophyll at both of 25 and 35?C. Spectral analysisof senescent leaves at 35?C in ,'-dipyridyl showed simultaneousbleaching the carotenoid and chlorophyll. (Received February 20, 1984; Accepted June 14, 1984)     

An {alpha}-Glucan from Suspension-Cultured Rice Cells

An -glucan was isolated from 11-day-old suspension-culturedrice cells by extraction with hot Na-phosphate buffer (pH 6.8).The -glucan had []_D=+234? (C = 0.14, in water) and its averagemolecular weight was estimated to be about 1.4 ? 10⁴, basedon elution characteristics on acalibrated Sepharose CL-6B column.Upon partial acid hydrolysis, the -glucan gave mainly malto-oligosaccharides.The maximum absorption of the iodine complex of the -glucanin the presence of Na₂SO₄ was at 470 nm. The results of hydrolysisby , ß- and iso-amylases and methylation analysisindicated that the isolated -glucan is a highly branched polysaccharidewith an average chain length of 9. The exterior and interiorchain lengths of the -glucan were calculated to be 5 and 3,respectively. (Received July 23, 1986; Accepted February 7, 1987)     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

Function of the {alpha} Subunit of Rice Heterotrimeric G Protein in Brassinosteroid Signaling

The subunit of plant heterotrimeric G proteins (G) plays pivotalroles in multiple aspects of development and responses to planthormones. Recently, several lines of evidence have shown thatG participates in brassinosteroid (BR) responses in Arabidopsisand rice plants. In this study, we conducted a comprehensiveanalysis of the roles of the rice G in the responses to BR usinga defective mutant of the G gene, T65d1. Decreased sensitivityto 24-epi-brassinolide (24-epiBL) in the T65d1 mutant was observedin many processes examined, e.g. in the inhibition of root growthand the promotion of coleoptile elongation. The T65d1 mutantalso showed similar phenotypes to those of BR-deficient mutants,such as the specifically shortened second internode and theconstitutive photomorphogenic growth phenotype under dark conditions.However, a negative feedback effect by 24-epiBL on the expressionof BR biosynthetic genes was observed in the T65d1 mutant, andthe levels of BR intermediates did not fluctuate in this mutant.To determine the epistatic relationship between the T65d1 mutantand d61-7, a weak allele of a rice BR receptor mutant, the twomutants were crossed. The T65d1/d61-7 double mutant showed noepistasis in the elongation inhibition of the internodes, theinternode elongation pattern, the leaf angle and the morphologicalabnormality of leaf, except for the vertical length of seedand the seed weight. Our results suggest that the rice G affectsthe BR signaling cascade but the G may not be a signaling moleculein BRI1-meditated perception/transduction.     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Pumpkin (Cucurbita sp.) seed globulin III. Comparison of subunit structures among seed globulins of various Cucurbita species and characterization of peptide components

Various Cucurbita seed globulins showed patterns similar toone another on SDS-gel electrophoresis, and ß bandsfor unreduced globulins and , ', and ' bands for reduced ones.On gel electrophoresis in 6 M urea, reduced globulin gave twoacidic and two basic bands. These corresponded to and ' chainsand ₁ and ₂ chains, respectively, identified by two-dimensionalurea-SDS gel electrophoresis. The compositions of the and ßsubunits were proposed. (Received September 8, 1977; )     

Ethylene Production by Cell-Free Extract of the Kudzu Strain of Pseudomonas syringae pv.phaseolicola

A cell-free ethylene-forming system of Pseudomonas syringaepv.phaseolicola (Kudzu strain) was characterized by its psychrophilictrait. Ethylene was most effectively produced from -ketoglutaricacid (-KG) at 0.5 mM followed by glutamate and then istidineat 5 to 10 mM. The presence of FeSO₄ was essential to the cell-freesystem. DTT and histidine greatly stimulated ethylene production;the latter could be substituted to some extent by its analogues.The optimum pH value and temperature for the ethylene-formingreactions were pH 7.0 and 25?C, respectively. Ethylene formationfrom -KG was inhibited in the presence of carbonates or organicacids of the TCA cycle, whereas that from glutamate was inhibitedin the presence of ammonium salts. Ethylene production from-keto--methylthiobutyric acid in the cell-free system was largelydependent on non-enzymical processes in the presence of DTTand FeSO₄. The ethylene-forming reactions were inhibited completelyby 1 mM n-propyl gallate and 1 mM p-chloromercuribenzoic acidand partly by coenzymes such as pyridoxal-1-phosphate, folicacid, and flavin mononucleotide at 5mM. The complete systemfor the highest ethylene production consisted of: 0.5 mM -KG,50 mM HEPES (pH 7.0), 5 mM DTT, 0.5 mM FeSO₄, and 10 mM histidine.The amount of ethylene produced in this system was equivalentto 40 to 50% of that produced by the living cells. (Received October 22, 1986; Accepted January 19, 1987)     

HORMONAL REGULATION ON THE INDUCTION OF {alpha}-AMYLASE ISOZYMES IN THE EMBRYO-LESS ENDOSPERM OF BARLEY

The -amylase induced by helminthosporol and gibberellic acidin the embryo-less endosperm of barley was separated into thethree fractions, 1, 2 and 3, by DEAE-cellulose column chromatography.A maximum amount of the 2. was induced by gibberellic acid andthat of the as by helminthosporol. After rechromatography, the2 induced by gibberellic acid and the as induced by helminthosporolshowed their respective single bands in an electrophoresis agargel zymogram. On the other hand, the ai induced by helminthosporoland gibberellic acid showed three bands. Dihydrohelminthosporic acid, a derivative of helminthosporol,induced the same isozymes as helminthosporol did. (Received May 8, 1967; )     

Brassinosteroid-Induced Bending of the Leaf Lamina of Dwarf Rice Seedlings: An Auxin-Mediated Phenomenon

A synthetic brassinosteroid (BR), 2,3,22ß,23ß-tetrahydroxy-24ß-methyl-B-homo7-oxa-5-cholestan-6-one, an isomer of the growth promoter brassinolide,when applied to seedlings of dwarf rice Oryza sativa var. Tan-ginbozuand Waito-C, induced a significant bending of the second leaflamina at 100 ng/plant and higher dosages. Promotion of thesecond leaf sheath elongation, the characteristic response ofdwarf rice varieties to gibberellins, was significantly butmodestly enhanced by BR at a dosage of 10,000 ng/plant, fiveorders of magnitude higher than the minimal dosage responseto GA₃. Gibberellin A₃ had no significant effect on the bendingof the second leaf lamina, nor did any synergism exist betweenBR and GA₃ in leaf lamina bending or leaf sheath elongation.Neither ethylene nor (2-chloroethyl)phosphonic acid (ethephon)caused the bending of the second leaf lamina, and neither synergizedthe BR effect. However, IAA and -naphthaleneacetic acid causedsignificant bending at 5,000 ng/plant, and both auxins significantlysynergized the effect of BR on the bending, IAA being effectiveat 500 ng/ plant in this regard. The antiauxins, 2,3,5-triiodobenzoicacid (TIBA) and -(p-chlorophenoxy)isobutyric acid (PCIB) completelynullified both the BR-induced bending and the BR$IAA-synergizedbending. The BR-induced bending response may thus be mediatedthrough endogenous auxin. (Received May 11, 1982; Accepted August 25, 1982)     

Inhibition of 1-Aminocyclopropane-l-carboxylic Acid Synthase Activity by Polyamines, Their Related Compounds and Metabolites of S-adenosylmethionine

Basic amino acids, monoamines, diamines and polyamines inhibitedthe activity of 1-aminocyclopropane-1-carboxylic acid (ACC)synthase extracted from wounded mesocarp tissue of winter squashfruit (Cucurbita maxima Duch.). Among the amines tested, polyamineswere highly effective, while the synthetic triamine, 1,8-diamino-4-aminomethyloctane,was an even stronger inhibitor than the polyamine spermine.Polyamines inhibited ACC synthase activity in a non-competitivemanner, while metabolic inhibitors such as aminoethoxyvinylglycineand aminooxyacetic acid inhibited ACC synthase activity competitively,showing much lower Ki values than those of polyamines. ACC synthaseactivity was also inhibited by intermediates of the methionine-recyclingpathway, 5'-methylthioadenosine and -keto--methylthiobutyricacid and by S-adenosylhomocysteine, a product of transmethylationof S-adenosylmethionine. It appears that polyamines not only inhibit ACC synthase activitybut also suppress the induction of the enzyme. However, unlikeprevious reports, polyamines did not inhibit in vivo ethyleneforming enzyme activity in the wounded mesocarp tissue. (Received October 24, 1985; Accepted January 10, 1986)     

Induction of Sexual Agglutinability by the Absorbed a Peptidyl Factor in a Mating Type Cells of Saccharomyces cerevisiae

The sexual agglutinability of inducible cells was induced byabsorbed pheromone in the absence of external a pheromone.The absorption needed no metabolic activity, but the inductiondid. This induction of sexual agglutinability by absorbed pheromonewas not accompanied or preceded by G1 arrest. The role of thebinding substance for pheromone in this induction is discussed. (Received November 13, 1980; Accepted December 19, 1980)     

Physiological detection of a binding substance for the agglutinability-inducing pheromone, {alpha} substance-I in Saccharomyces cerevisiae

We found a substance which binds to substance-I to inactivatethe biological activity of the pheromone to induce sexual agglutinabilityof a mating type cells. Both living and boiled cells of thea mating type had the substance-I-absorbing action. The absorbingaction of living cells was detected almost equally at both 0and 28?C. Cell extract of the a mating type showed the substance-I-inactivatingaction. The biological activity of substance-I inactivatedby shaking with cell-free culture medium of the a mating typewas recovered by heating at 100?C, which destroyed the inactivatingaction of the culture medium with little effect on the substance-Iactivity, indicating that a substance in the culture mediuminactivated substance-I by binding to it. This is supportedby the fact that the inactivating action completely stoppedin 30 min, leaving a considerable amount of active substance-I,when the concentration of the inactivating substance was lowcompared with that of substance-I. The ability to produce thebinding substance as specific to the a mating type. The bindingsubstance was different from the a agglutination substance responsiblefor sexual agglutination. ² On leave from Osaka City University. (Received April 6, 1977; )     

Purification and properties of soluble cytochrome b-560 from spinach leaves

A b-type cytochrome having an -band at 560 nm was isolated fromspinach leaves (Spinacia oleracea). A method is described forpreparing this cytochrome, cytochrome b-560 (spinach), in apurified state. The cytochrome has, in its reduced state, absorption bands at560 nm (), 530 nm (ß) and 427 nm (); and in the oxidizedstate at 562 nm (), 529 nm (ß) and 417 nm (). Thepyridine ferro-haemochrome prepared from cytochrome b-560 hadan -band at 556.5 nm, indicating the protohaem-nature of theprosthetic group. The cytochrome has an oxidation-reduction potential (E'⁰) of+0.13V at pH 7.0, as measured using the ferri-ferro oxalate system. The cytochrome is rapidly reduced on illumination with red orfar-red light in the presence of spinach chloroplasts and isoxidized at a slower rate in the dark. This photoreduction isinhibited by 1x10^–6 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The molecular weight of the cytochrome is 30,000 asestimated by the dextran gel filtration method. (Received December 3, 1971; )