Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

Effect of CO2 Concentration on Growth, Carbon Distribution and Loss of Carbon from the Roots of Maize

The effects of three ranges of CO₂ concentration on growth,carbon distribution and loss of carbon from the roots of maizegrown for 14 d and 28 d with shoots in constant specific activity¹⁴CO₂ are described. Increasing concentrations of CO₂ led toenhancement of plant growth with the relative growth rate (RGR)of the roots affected more than the RGR of the shoots. Between16% and 21% of total net fixed carbon (defined as ¹⁴C retainedin the plant plus ¹⁴C lost from the root) was lost from theroots at all CO₂ concentrations at all times but the amountsof carbon lost per unit weight of plant decreased with time.Possible mechanisms to account for these observations are discussed. Key words: Growth, Roots, Carbon loss, [CO₂]     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)     

Purification and Properties of L-Arginine Decarboxylase of Evernia prunastri

An L-arginine decarboxylase was isolated from Evernia prunastrithallus. The enzyme was purified about 117-fold and showed apH optimum of 7.1 and a temperature optimum at 26°C. Itsmolecular weight was estimated as 300,000. The Evernia argininedecarboxylase was significantly inhibited by L-ornithine, ureaand putrescine. The K_m for L-arginine was about 12.5 mM. (Received March 9, 1981; Accepted June 26, 1981)     

Effect of mineral deficiency on amine formation in higher plants

Potassium deficiency caused putrescine accumulation in the leaves of barley, radish, pea, bean and spinach plants. Magnesium deficiency caused putrescine accumulation in barley, pea and bean leaves, and also in the leaves of older radish plants. In young radish plants less putrescine was found in magnesium deficiency, and in spinach magnesium deficiency was without effect on putrescine levels. Putrescine content may be a useful guide to the mineral status of legumes, since accumulation of this amine may be detected before deficiency symptoms appear. Radioactivity from l-arginine-[U-¹⁴C] fed to barley seedlings was detected in agmatine within 2 hr, and probably also in the hordatines after 24 hr, feeding. After 2 hr the label in the agmatine was greatest in the potassium-deficient plants, but after 24 hr the level declined to that found in the agmatine of the leaves of the magnesium-deficient and control seedlings. The rate of putrescine formation was high in both potassium and magnesium deficiency. Incorporation of radioactivity in spermidine and spermine on feeding putrescine-[1,4-¹⁴C] to barley seedlings was estimated in the dansylated amines after separation by TLC. Activity was higher in spermidine and lower in spermine in the potassium-deficient plants than in the controls. The spermidine/spermine ratio declined on excision of barley leaves.     

Modulated Degradation of Ribulose Bisphosphate Carboxylase in Leaves on Top-Pruned Shoots of the Mulberry Tree (Morus alba L.)

Yamashita, T. 1987. Modulated degradation of ribulose ftisphosphatecarboxylase in leaves on top-pruned shoots of the mulberry tree(Morus alba L.).—J. exp. Bot. 38: 1957–1964. The effects of pruning shoot tops on the synthesis and degradationof ribulose 1,5–Wsphosphate carboxylase (RuBPCase) inleaves on remaining shoots were investigated in mulberry trees.Leucine labelled with ¹⁴C was fed to leaf discs from field-grownmulberry trees and ¹⁴C incorporation into RuBPCase was examined.Proportion of ¹⁴C in RuBPCase to leucine–¹⁴C absorbedby leaf discs was remarkably lowered by top-pruning, thoughoccasionally a slight increase was observed soon after pruning.Yet RuBPCase content in leaves on top-pruned shoots became progressivelyhigher than that in leaves on intact shoots. Changes in ¹⁴Cin Ru1BPCase in leaves of mulberry saplings previously fed ¹⁴CO₂were followed. Following ¹⁴CO₂ feeding, the attainment of themaximal level of ₁₄C in RuBPCase was retarded by top-pruning.The highest level of ₁₄C in RuBPCase was maintained in leaveson top-pruned shoots but decreased in leaves on intact shoots.Specific radioactivity in RuBPCase continued to increase inleaves on top-pruned shoots even after attaining a maximum levelin the control leaves. These facts suggest that the increasein RuBPCase by top-pruning results from a cessation of its degradationfor the remobilization of nitrogen for newly developing leaveson shoot tops. Key words: RuBP carboxylase, shoot pruning, mulberry (Morus alba)     

An Examination of the Value of 14C-urea as a Source of 14CO2 for Studies of Assimilate Distribution

Twenty-four hours after leaf 3 of a plant of Lolium multiflorumLam, was supplied with a droplet of ¹⁴C-urea and the plant enclosedin a polyethylene bag with an untreated plant, there were significantamounts of radiocarbon recovered from the untreated plant. Theleaf treated with ¹⁴C-urea was the major source of ¹⁴C leakagebut significant losses were also recorded from other parts ofthe plant. Reducing the humidity within the bag decreased theamount of ¹⁴CO₂ which escaped. Losses of radiocarbon from CO₂-treated plants were very low compared with those from urea-treatedplants but the pattern of assimilate distribution within thetwo types of plants was very similar. The possible causes ofthese effects are considered and the usefulness of ¹⁴C-ureaas a source of ¹⁴CO₂ discussed.     

The Effects of Gibberellic Acid on Organelle Biogenesis in the Endosperm of Germinating Castor Bean Seeds

Mature seeds of castor bean (Ricinus communis L.) were imbibedin tap water or 0.3 mM GA₃, planted in vermiculite moistenedwith tap water or 0.3 mM GA₃, and incubated at 32 ?C. Duringthe course of germination, GA₃ promoted marked increases inthe activities of the glyoxysomal marker enzyme isocitrate lyaseand certain mitochondrial marker enzymes, but did not affectthe ER marker enzyme choline phosphotransferase. Glyoxysomaland ER protein and phospholipid were not increased in amountby GA₃, whereas mitochondrial protein and phospholipid were.SDS-polyacrylamide gels of the glyoxysomal matrix polypeptidesfrom GA₃-treated beans exhibited two polypeptides additionalto those found to be common to both GA₃-treated and controltissue. Incorporation of CDP-(methyl ¹⁴C)-choline into intactendosperm tissue and the distribution amongst the glyoxysomes,mitochondria, and ER of newly synthesized phosphatidyl-(methyl¹⁴C)-choline was unchanged by GA₃.     

Effects of Gibberellic Acid on the Activation, Synthesis, and Release of Acid Phosphatase in Barley Seed

Acid phosphatase activity was present in unimbibed barley seed,but rose during incubation of embryoless half-seeds and isolatedaleurone layers, and was further increased by 10^–6 M gibberellicacid (GA₃). Release of total acid phosphatase activity fromhalf-seeds and aleurone layers was markedly enhanced by GA₃.Inhibitor studies with cycloheximide and actinomycin D suggestedthat de novo synthesis of acid phosphatase occurred followingimbibition. Gel nitration, electrophoresis, and [¹⁴C]leucineincorporation studies revealed that a single molecular formof acid phosphatase was present in dry seed, whereas on incubationtwo further forms arose. A proportion of the three molecularforms of the enzyme was synthesized de novo. Gibberellic acidstimulated activation, but not de novo synthesis, of all threemolecular forms of acid phosphatase. Although a small amountof one of the molecular forms was secreted in the absence ofGA₃, the presence of gibberellin greatly increased secretionof the same form of acid phosphatase.     

Photosynthetic carbon metabolism in wild, primitive and cultivated forms of wheat at three levels of ploidy: Role of the glycolate pathway

¹⁴CO₂ assimilation was studied with diploid, tetraploid, hexaploidspecies of the genera Triticum and their wild relatives Aegilops.Attached mature leaves of 3–4 weekold plants were allowedto undergo photosynthesis under air at ambient temperature.The pattern of distribution of ¹⁴C was notably similar in Triticumand Aegilops species whatever the level of ploidy. Sucrose wasthe sink for photosynthetic carbon. ¹⁴C for sucrose synthesis was supplied either through the glycolatepathway by glycolate, the product of the photorespiration orby the Calvin cycle intermediates exported into the cytoplasm.Depending on the species, the glycolate pathway provided 40to 75%of the sucrose ¹⁴C. The higher labeling of sucrose was associated with the greaterparticipation of the glycolate pathway in the wild diploid (DD)A. squarrosa and in the cultivated hexaploid (AABBDD) T. aestivum.The results suggest that the expression of the male D genomeis dominant over the female AB genome in T. aestivum. In T. aestivum under ambient conditions lowering (low temperature)or hindering (1% O₂ ) photorespiration, sucrose labeling decreased,but serine and glycine labeling was favoured. We propose thatin wheat leaves, the role of photorespiration is to drain artof the carbon exported from the chloroplast as glycolate, towardssucrose synthesis. (Received March 16, 1979; )     

Glycollate Formation during the Photorespiration of Acetate by Chlorella

WhenChlorella pyrenoidosa photoassimilates ³H-¹⁴C-acetate theglycollic acid formed shows a high ³H/¹⁴C ratio, the only othercompounds showing similar ratios being glycerate and serine.The ³H/¹⁴C ratio of glycollate was unaffected by the TCA cycleinhibitors MFA, diethylmalonate and arsenite showing that ³Hin glycollate does not result from the oxidation of acetatevia the TCA cycle, the resulting NADP³H₂ or NAD³H₂ being usedfor the reduction of the glycollate precursor. Although DCMUdecreased the ³H/¹⁴C ratio, complete inhibition of glycollatelabelling was not observed with 10^–6 M DCMU, at whichconcentration complete inhibition of the Hill reaction is achieved.Although the ³H/¹⁴C ratio was unaltered, total dpm of both ¹⁴Cand ³H in glycollate were increased by INH. The ³H/¹⁴C ratiosof glycerate and serine were decreased by INH, as were the totaldpm of ³H and ¹⁴C incorporated into these compounds. Thus, INHinhibits the further metabolism of glycollate to glycerate andserine. The effect of INH on incorporation of ¹⁴C-I-acetateinto various cell fractions was investigated. The incorporationof ¹⁴C into polysaccharide and lipid was decreased, while theincorporation of ¹⁴C into the water-soluble fraction of cellsand therelease of ¹⁴CO₂ were little affected. Although glycollicacid was an early product of acetate photoassimilation in Chlorellapyrenoidosa, glycollate excretion does not take place undera wide range of environmental conditions shown to favour glycollateexcretion by other algae. However, small amounts of labelledglycollate were detected in the supernatant from the cells duringthe photoassimilation of ³H-¹⁴C-acetate, but this glycollatedid not show the high ³H/¹⁴C ratio of glycollate present withinthe cell. The failure of Chlorella pyrenoidosa to excrete appreciableamounts of glycollate when photoassimilating acetate or carbondioxide was considered to result from the presence of glycollateoxidase (EC 1.1.3.1 [EC] ) which allowed the further metabolism ofglycollate. Besides glycollate oxidase, glyoxylate reductasewas also demonstrated in Chlorella pyrenoidosa so that glycollatecould function in hydrogen transfer during the photoassimilationof acetate.     

Glycolate synthesis in a C3 C4 and intermediate photosynthetic plant type

Excised leaves of a C₃-photosynthetic type, Hordeum vulgare,a C₄-type, Panicum miliaceum, and an intermediate-type, Panicummilioides, were allowed to take up through their cut ends a1 mM solution of butyl hydroxybutynoate (BHB), an irreversibleinactivator of glycolate oxidase. After 30 to 60 min in BHB,extractable glycolate oxidase activity could not be detectedin the distal quarter of the leaf blades. Following this pretreatment,recovery of ¹⁴C-glycolate from ¹⁴CO₂ incorporated in a 10 minperiod was nearly maximal for each of the three plant types.Labeled glycolate was 51% of the total ¹⁴CO₂ incorporated forthe C₃-species, 36% for the intermediate-species, and 27% forthe C₄-species Increased labeling of glycolate was compensatedfor primarily by decreased labeling of the neutral and basicfractions for the C₃ and intermediate-type species. In the C₄-type,label decreased primarily in the neutral and insoluble fractions,but increased in the basic fraction. A lower rate of glycolatesynthesis is indicative of a lower rate of photorespirationand consistent with a lower O₂/CO₂ ratio present in the bundle-sheathcells of C₄-plants. We conclude that both decreased glycolatesynthesis and the refixation of photorespiratory-released CO₂are important in maintaining a lower rate of photorespirationin C₄-plants compared to C₃ plants. Intermediate glycolate synthesisin Panicum milioldes is consistent with its intermediate levelof O₂ inhibition of photosynthesis and intermediate rate ofphotorespiration. (Received May 6, 1978; )     

Effects of Atmospheric O3 on Azolla-Anabaena Symbiosis

Cultures of water fern Azolla pinnata R. Br. exposed for 1 weekto either 30, 50 or 80 nl l^-1 O₃ showed significant reductionsin rates of growth and N₂ fixation, and had fewer heterocysts.Although the levels of glutamine synthetase (GS) and glutamatedehydrogenase (GDH) activity were decreased by low concentrationsof O₃ exposures (30 or 50 nl l^-1), significant increases inlevels of the same enzymes were caused by higher concentrationsof O₃ (80 nl l^-1). Increased levels of total protein, polyamines(putrescine and spermidine), and the xanthophyll-cycle precursorof abscisic acid (ABA), violaxanthin, were also found with higherlevels of O₃ (80 nl l^-1). Levels of ABA itself were significantlyincreased by low level O₃ fumigation (30 nl l^-1) but significantlydecreased by exposure to 80 nl l^-1 O₃. This may indicate thathigher levels of atmospheric O₃ inhibit the final stages ofABA biosynthesis from violaxanthin.Copyright 1994, 1999 AcademicPress Abscisic acid, nitrogen assimilation, nitrogen fixation, ozone pollution, polyamines, violaxanthin     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)     

Carbon Loss from the Roots of Forage Rape (Brassica napus L.) Seedlings Following Pulse-labelling with 14CO2

The loss of organic material from the roots of forage rape (Brassicanapus L.,) was studied by pulse-labelling 25-d-old non-sterilesand-grown plants with ¹⁴CO₂. The distribution of ¹⁴C withinthe plant was measured at 0, 6 and 13 d after labelling whilst¹⁴ C accumulating in the root-zone was measured at more frequentintervals. The rates of ¹⁴C release into the rhizosphere, andloss of ¹⁴CO₂ from the rhizosphere were also determined. Thesedata were used to estimate the accumulative loss of ¹⁴C fromroots and loss respiratory ¹⁴CO₂ from both roots and associatedmicro-organisms. Approximately 17-19% of fixed ¹⁴CO₂ was translocatedto the roots over 2 weeks, of which 30-34% was released intothe rhizosphere, and 23-24% was respired by the roots as ¹⁴CO₂. Of the ¹⁴C released into the rhizosphere, between 35-51%was assimilated and respired by rhizosphere micro-organisms.Copyright1993, 1999 Academic Press Brassica napus L., carbon loss, carbon partitioning, microbial nutrition, microbial respiration, forage rape, pulse-labelling, rhizodeposition, root respiration, sand culture