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)     

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; )     

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.     

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; )     

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)     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

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)     

Polyamine Metabolism in the Roots of Phaseolus vulgaris. Interaction of the Inhibitors of Polyamine Biosynthesis with Putrescine in Growth and Polyamine Biosynthesis

The effects of the inhibitors of polyamine biosynthesis, canavanineand -methyl ornithine on growth, the activities of argininedecarboxylase (EC 4.1.1.19 [EC] ) and ornithine decarboxylase (EC4.1.1.17 [EC] ) and on polyamine content were examined in two differentgrowth regions of Phaseolus vulgaris L. cv. Taylor's Horticulturalroots. Separately, in the same manner, in the same bean rootsystem exogenous putrescine effect and the interaction of canavaninewith putrescine were determined. The arginine and ornithine decarboxylase activities found inroot apex were high where cell division activity was highest.Polyamine (putrescine and spermine) content did not correlatewith these activities, but polyamine level was high in the rootbase where cell elongation is the main process. The arginineanalogue, canavanine, inhibited arginine decayboxylase activityand polymine liters. Putrescine partially reversed the canavanineinhibition of root growth as well as arginine decarboxylaseactivity and polyamine content. Similarly -methyl ornithineslightly inhibited the root length and ornithine decarboxylaseactivity in the root apex. Besides, exogenous putrescine didnot effect significantly the endogenous polyamine titers. Theseresults reinforce the growing connection between polyaminesand the rates of cell devision in the roots of bean plants.Separately, arginine decarboxylase is the main enzyme in thebean roots. (Received November 10, 1986; Accepted March 3, 1987)     

Improvement of the indolo-{alpha}-pyrone fluorescence method for quantitative determination of endogenous indole-3-acetic acid in lettuce seedlings

The indolo--pyrone fluorescence method was modified to measurethe endogenous amount of indole-3-acetic acid (IAA) in lettuceseedlings, because impurities in lettuce extracts made the indolo--pyronesolution turbid, if the reaction of the extracts with aceticanhydride to give indolo--pyrone was disrupted with 3 ml distilledwater. This turbid solution caused light scattering, resultingin overestimating the IAA amount in lettuce seedlings. We could,however, obtain a clear indolo--pyrone solution by disruptingthe reaction with 0.2 ml distilled water and 2.8 ml acetic acidin place of 3 ml distilled water. We examined the reliabilityof the new method and found that the use of acetic acid substantiallyslowed down the rate of indolo--pyrone breakdown and did notdecrease the sensitivity of the method. The effect of gibberellicacid on the endogenous amount of IAA in lettuce seedlings wasalso examined. ¹ One of the authors (S. K.) received for this work a grantfrom Osaka City University to study abroad. (Received January 11, 1977; )     

{alpha}-Amylase synthesis by a cell free system and plant growth substances

A microsomal preparation from the aleurone layer pre-treatedwith GA or H-ol had the ability to synthesize -amylase whenit was incubated with an appropriate medium. -Amylase synthesiswas inhibited by the addition of p-fluorophenylalanine or bypre-treatment with RNase. The synthesized -amylase was separatedinto three isozymes by disc-electrophoresis (Received July 25, 1970; )     

Promotion and inhibition of {alpha}-amylase production in barley endosperm by cyclic 3',5'-adenosine monophosphate and adenosine diphosphate

The possibility that gibberellin-induced -amylase synthesisin barley endosperm might be mediated by cyclic-3',5'-adenosinemonophosphate (3',5'-AMP) was examined. Promotion of -amylasesynthesis by 3',5'-AMP (5 mM) was observed in the absence ofgibberellic acid (GA₃) and in combination with GA₃ at concentrationsbelow 2 mµM. When combined with gibberellin at concentrationsabove 2 mµM, however, 3',5'-AMP reduced the amount of-amylase obtained. The cyclic nucleotide showed slight activityat concentrations as low as 0.05 mM. These promotions were shownto be due to increased synthesis of -amylase rather than toan increased secretion of the enzyme. Of a variety of adeninecompounds and nucleoside diphosphates tested only 3',5'-AMPand adenosine diphosphate (ADP) induced -amylase synthesis.Longer incubation times were necessary to obtain maximal -amylaseinduction with the nucleotides than with GA₃. ADP and 3',5'-AMPwere about one third and one fifth as active, respectively,as GA₃ in promoting -amylase synthesis, although GA₃ was morethan 10⁷ times more effective. AMO-1618 did not inhibit theaction of the nucleotides and methanolic extracts of the nucleotidesshowed no gibberellin-like activity. Both nucleotides were synergisticwith GA₃ in overcoming the inhibitory effects of acetate andcitrate buffers on -amylase synthesis. (Received February 24, 1969; )