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)     

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)     

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)     

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)     

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; )     

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; )     

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.     

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)     

Senescence of Rice Leaves XXX Levels of Endogenous Polyamines and Dark-Induced Senescence of Rice Leaves

The role of endogenous polyamines in the control of dark-inducedsenescence of detached rice leaves was investigated by quantitatinglevels of various polyamines by HPLC. Putrescine, spermidineand spermine were all present throughout senescence. Neithercadaverine nor 1,3-diaminopropane was detected. During dark-inducedsenescence, there was a marked decrease in levels of putrescineand an increase in those of spermidine and spermine. The rateof production of ethylene increased markedly upon excision ofleaves. -Difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO) caused a reduction in levels of putrescine, yet had noeffect on levels of spermidine and spermine. Neither DFMA norDFMO had any effect on senescence or on the production of ethylene.Treatment with dicyclohexylamine (DCH) and methylglyoxal bis-(guanylhydrazone)(MGBG) reduced levels of spermine and increased those of putrescinein detached leaves. After treatment with DCH or MGBG, both senescenceand the production of ethylene were significantly promoted.The current results suggest that endogenous polyamines may notplay a significant role in the control of dark-induced senescenceof rice leaves. This conclusion is supported by the furtherobservations that (a) benzyladenine, which is known to retardsenescence, decreased levels of putrescine but had no effecton those of spermidine and spermine; and (b) ABA, which promotedsenescence, increased levels of putrescine and had no effecton those of spermidine and spermine. (Received March 30, 1991; Accepted June 27, 1991)     

ON THE EFFECTS OF PLANT EXTRACTS ON THE {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM

Effects of ethanol extracts obtained from several plant materialson -amylase activity in rice endosperm were examined. Promotingeffect on the -amylase activity was estimated in terms of gibberellinA₃ equivalence using the embryoless endosperm. Inhibiting effectwas examined using intact seed. On chromatography of the extractfrom Prunus, the zones showing promotion of the -amylase activityalso exhibited the activity for the elongation of the leaf sheathof rice seedling. But this was not the case with the extractsfrom Pharbitis, Allium and Lupinus. (Received April 13, 1966; )     

A Mutation Leading to Constitutive Expression of High Sexual Activities in the Yeast Saccharomyces cerevisiae

An a mating type mutant of the yeast Saccharomyces cerevisiaewhich expressed high sexual activities during vegetative growthwas isolated and characterized. Its constitutive sexual agglutinabilitywas higher than the sexual agglutinability of its parental straininduced by pheromone. It produced a pheromone and -pheromone-inactivatingsubstances in larger amounts than its parental strain. It alsoproduced large pear-shaped cells (shmooed cells) without pheromone,was more sensitive to pheromone, and grew vegetatively moreslowly than its parental strain. When the mutant was crossedto a wild type strain isogenic with the parental strain, amating type segregants with high constitutive sexual agglutinabilityshowed self-shmooing. However, in a mating type segregants self-shmooingwas not observed regardless of the degree of their sexual agglutinability.The cross between a and segregants, both of which carried themutation, had higher frequency of zygote formation than thecrosses between a and cells one of which or both of which wereof wild type. (Received September 9, 1985; Accepted February 8, 1986)     

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; )     

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)     

{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; )     

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; )     

Discovery of Natural Photosynthesis using Zn-Containing Bacteriochlorophyll in an Aerobic Bacterium Acidiphilium rubrum

We discovered natural photosynthesis using Zn-containing bacteriochlorophyll in an acidophilic bacterium Acidiphilium rubrum. Chemical analysisof the cell extracts gave a 13 : 2 :1 molar ratio of Zn-bacteriochlorophyll : Mg-bacteriochlorophyll : bacteriopheophytin . Most of thepigments are associated with fully active reaction center andlight-harvesting complexes analogous to those in purple photosyntheticbacteria. The finding indicates an unexpectedly wide variabilityof photosynthesis. ⁷Present address: Department of Ecological Engineering, ToyohashiUniversity of Technology, Tenpaku-cho, Toyohashi, 441 Japan     

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)