Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Effect of Nitrogen on Nitrate Reductase Activity in the Nodules and Leaves of Summer Moong (Vigna radiata)

The relation of the in vivo nitrate reductase (NR) activityto growth period was studied in the nodules and the leaves ofthe summer moong (Vigna radiata). The maximum NR activity wasobserved 31 days after sowing (DAS) in the leaves and 28 DASin the case of the nodules. In a pot experiment, the effectof the various nitrogen concentrations, namely 0, 3, 6, 9 and12 mg kg^–1 was studied on NR activity at three growthstages. The maximum NR activity was observed at 6 mg kg^–1N during the pre-flowering stage (26 DAS). Though the noduleshave higher NR activity, its expression was limited by substrateavailability. The NR activity in the leaf could be used as anindex of NR activity in the nodules. Nitrate reductase, nitrogen, nitrate, moong, Vigna radiata     

Properties of a Large Complex with NADH Dehydrogenase Activity from Barley Thylakoids

When assays for NAD(P)H-ferricyanide oxidoreductases were performed,activities specific for NADH (0.23 unit (mg protein)^–1)and NADPH (0.68 unit (mg protein)^–1) were detected inchloroplasts isolated from leaves of barley (Hordeum vulgareL.). Activities of chloroplast NADH- and NADPH-ferricyanideoxidoreductase were 5-fold and 25-fold higher, respectively,than the maximum activity that could be attributed to mitochondrialcontamination. Moreover, most of the chloroplast NADH-ferricyanideoxidoreductase (60 to 80%) was solubilized by deoxycholate (DOC)from thylakoids as a single, high-molecular-mass complex thatwas distinguishable from the mitochondrial complex by its lowerelectrophoretic mobility in 3% polyacrylamide, as revealed byreduction of nitro blue tetrazolium (NBT) in the presence ofNADH or NADPH on gels after electrophoresis. The stroma yieldeda single band of a dehydrogenase (66 kDa) that used NADH asits electron donor. Several NADPH-dependent activities weredetected after electrophoresis of the stromal fraction. Moreover,chloroplast-specific activities could be distinguished frommitochondrial activities on the basis of the specificity ofthe donor and the acceptor of electrons, the dependence of theactivities on pH, and the sensitivity to various inhibitors.K_m values for NADH (26 µM) and NADPH (75 µM) werein the same range as those of mitochondrial activities. Mostof the NADPH-dependent activity probably corresponds to thechloroplast ferredoxin-NADP⁺ oxidoreductase. The possibilityis discussed that thylakoid NADH dehydrogenase(s) might be theproduct of chloroplast ndh genes and that this activity is involvedin chlororespiration. (Received April 25, 1994; Accepted December 5, 1994)     

Nitrate Reduction by Cassava

Nitrate reductase (NR) was assayed in vivo in cassava (Manihotesculenta Crantz). Activity in the leaves ranged from 0 to 2.51µmole of NO₃^– reduced g^–1 h^–1, withno activity in the younger leaves (leaf 1 on top). NR activitywas localized in the sides and toward the tip of the lobes ofthe leaf. (Received December 10, 1985; Accepted April 8, 1986)     

In vivo Nitrate Reductase Activity in Barley (Hordeum vulgare L.) during Ear Development

Experiments were conducted during the 1974–75 and 1975–76winter season with the barley (Hordeum vulgare L.) cultivarJyoti. From amongst the various plant parts, the flag leaf bladehad higher in vivo nitrate reductase (NR) activity than thelower two leaf blades, glumes, and grains. However, the potentialof a plant part to reduce NO₃^– is a function of its freshweight and the NR per unit fresh weight. On this basis, thesecond and third leaf blades could reduce more NO₃^– thanthe flag leaf blade. N fertilizer application resulted in enhancementof the activity of the leaf blades alone. N fertilizer appliedduring the reproductive phase was taken up and assimilated bythe various plant parts. The studies suggest that, even whenthe fertilizer is applied at optimum levels for obtaining maximumyields, the upper leaf blades have sub-optimal NR activity andthat there is a likelihood of either a preferential flow ofNO₃^– to the leaf blades or transnational barriers to NO₃^–movement to the ear.     

Photosynthetic Nitrogen Metabolism in High and Low CO2-adapted Scenedesmus: II. EFFECT OF AMMONIUM AND METHIONINE SULPHOXIMINE ON NITRATE UTILIZATION

Larsson, C.-M., Larsson, M. and Guerrero, M. G. 1985. Photosyntheticnitrogen metabolism in high and low CO²-adapted Scenedesmus.II. Effect of ammonium and methionine sulphoximine on nitrateutilization.—J. exp. Bot. 36: 1387–1395 In 3% CO²-grown Scenedesmus obtusiusculus Chod. utilizing NO₃^–J as the N source, NH₄⁺ addition caused a prompt inhibitionof NO₃^– utilization. Nitrate reductase (NR) activity declinedrapidly in response to the presence of NO₄⁺, but the cessationof NO₃^– utilization was too rapid to be accounted forby the loss in NR activity. The first site of NO₄⁺ inhibitionin these cells seems to be the entrance of NO₃^– into thecells. Upon exhaustion of NO₄⁺ from the medium, NO₃^– utilizationwas rapidly restored and NR activity increased. Air-grown cellswere much less sensitive to the effect of NO₄⁺, more than 30min being required for added NO₄⁺ to cause complete inhibitionof NO₃^– utilization. In these cells, NO₃^– uptakeand NR activity decreased in parallel in response to NO₄⁺ addition.In 3% CO²-grown cells simultaneously subjected to NO₄⁺ and air-levelof CO², NO₄⁺ initially inhibited NO₃^– utilization completely,but a slight recovery took place after approximately 20 min The glutamine synthetase (GS) inhibitor L-methionine D, L-sulphoximine(MSO) behaved as a potent inhibitor of NO₃^– uptake in3% CO²-grown cells, but had considerably less effect in air-growncells, although the time-course of the MSO-induced inhibitionof GS was the same in both cases Key words: Ammonium, nitrate utilization, Scenedesmus     

Nitrogen Economy of the Main Shoot of Field-Grown Barley (Hordeum vulgare L.): II. IN VIVO NITRATE REDUCTASE ACTIVITY DURING GROWTH

The method for assay of in vivo nitrate reductase (NR) activitywas standardized for barley (Hordeum vulgare L.). NR activitywas determined in the various organs of the main shoot of field-grownJyoti barley at 40 kg N ha_–1. Total nitrate reductaseactivity (TNRA) of each organ for the period it was metabolicallyactive was calculated. The NR activity was highest in the laminae,followed by the sheaths, reproductive organs; and internodes.The NR activity was high in the first-formed laminae and itshowed a decline in the ones formed subsequently. The valuesvaried from 43.2 ± 4.33 to 7.2 ± 1.49 µmolNO^–₃ reduced g^–1 dry wt. h^–1. Maximum TNRAin the laminae, sheath, and internodes was at 49, 84, and 84–93d after sowing, respectively. The TNRA of the main shoot asa whole showed three peaks, one around 49 d, a second around63 d, and a third around 84 d after sowing. Correlation coefficient(r) between NR and NO3 concentration was highly significantin the laminae and sheath viz. 0.76^*** and 0.62^***, respectively.The results are discussed in relation to alteration in managementpractices to maximize nitrate assimilatory activity and theamount of reduced N harvested.     

Inverse Correlation of Thiol Proteinase with Nitrate Reductase Activities in Barley Leaves

Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml^–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml^–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)     

Isolation, Purification and Some Properties of Cytochrome c-551 from Chromatium vinosum

Cytochrome c-551 was isolated and purified from a photosyntheticbacterium Chromatium vinosum by ammonium sulfate fractionation,ion-exchange chromatography and gel filtration. The cytochromehad absorption maxima at 280, 407 and 523–524 nm in theoxidized form, and 416, 521 and 549.5 nm in the reduced form.The reduced-minusoxidized difference millimolar absorption coefficientwas 9.90 mM^–1cm^–1 for the wavelength pair, 550.5minus 540 nm. The molecular weight of the cytochrome was 16,000by gel filtration on Sephadex G-100 and 15,500 by sodium dodecylsulfate-polyacrylamidegel electrophoresis. The midpoint redox potential was +240 mVat pH 8.0. Cytochrome c-551 was released from bacterial cells when spheroplastswere produced but EDTA and lysozyme treatments. The releasedcytochrome had the same properties as those of the cytochromepreparation obtained by disruption of cells through a Frenchpressure cell. This confirms the earlier suggestion that cytochromec-551 is located in the periplasmic space of cells. (Received August 21, 1982; Accepted October 28, 1982)     

Recruitment of NADH shuttling in pressure-overloaded and hypertrophic rat hearts

Glucose metabolism in the heart requires oxidation of cytosolic NADH from glycolysis. This study examines shuttling reducing equivalents from the cytosol to the mitochondria via the activity and expression of the oxoglutarate-malate carrier (OMC) in rat hearts subjected to 2 wk (Hyp2, n = 6) and 10 wk (Hyp10, n = 8) of pressure overload hypertrophy vs. that of sham-operated rats (Sham2, n = 6; and Sham10, n = 7). Moderate aortic banding produced increased atrial natriuretic factor (ANF) mRNA expression at 2 and 10 wk, but only at 10 wk did hearts develop compensatory hypertrophy (33% increase, P < 0.05). Isolated hearts were perfused with the short-chain fatty acid [2,4-¹³C₂]butyrate (2 mM) and glucose (5 mM) to enable dynamic-mode ¹³C NMR of intermediate exchange across OMC. OMC flux increased before the development of hypertrophy: Hyp2 = 9.6 ± 2.1 vs. Sham2 = 3.7 ± 1.2 µM·min^–1·g dry wt^–1, providing an increased contribution of cytosolic NADH to energy synthesis in the mitochondria. With compensatory hypertrophy, OMC flux returned to normal: Hyp10 = 3.9 ± 1.7 vs. Sham10 = 3.8 ± 1.2 µM·g^–1·min^–1. Despite changes in activity, no differences in OMC expression occurred between Hyp and Sham groups. Elevated OMC flux represented augmented cytosolic NADH shuttling, coupled to increased nonoxidative glycolysis, in response to hypertrophic stimulus. However, development of compensatory hypertrophy moderated the pressure-induced elevation in OMC flux, which returned to control levels. The findings indicate that the challenge of pressure overload increases cytosolic redox state and its contribution to mitochondrial oxidation but that hypertrophy, before decompensation, alleviates this stress response. malate-aspartate shuttle; redox state; hypertrophy     

Effects of Cytokinin and Several Inorganic Cations on the Polyamine Content of Lettuce Cotyledons

Kinetin (4.7 x 10^–5 M) and 6-benzyladenine (2.22 x 10^–5M) were found to increase ca. 2-fold the putrescine contentin cotyledons of lettuce (Lactuca sativa L.) seedlings grownfor 3 days under fluorescent light. On the other hand, severalinorganic ions (K⁺, Na⁺, Ga⁺⁺, Mg⁺⁺) at a concentration of 3x 10^–2 M reduced the putrescine content. The combinationof kinetin with one of several inorganic ions at the same levelmarkedly increased the spermine content, but the putrescinecontent decreased; calcium and magnesium ions were less effective.The physiological significance of these findings is discussed. (Received July 4, 1982; Accepted November 6, 1982)     

Effect of Chelating Agents and Metal Ions on Growth and Fertility in the Male Clones of the Moss Microdus brasiliensis (Dub.) Ther

The male gametophores of Microdus brasiliensis become fertileafter 48 d on basal medium. EDDH A increases gametophore numberand percentage of fertile gametophores at lower concentrations(10^–8-10^–6 mol dm^–3), whereas EDTA enhancesboth the responses at all levels (10^–8-10^–4 moldm^–3). Their iron salts increase gametophore number aswell as the number of fertile gametophores, and the latter effectis more striking. The number of antheridia per head also increaseswith Fe-EDTA, and at higher concentrations antheridia are induced4 d earlier. EDTA and Fe-EDTA-stimulated antheridia] formationis associated with a corresponding increase in endogenous iron.Copper content increases only at higher levels of EDTA and Fe-EDTA,and there is no correlation with the antheridial induction response.Salicylic acid increases the number of gametophores and thepercentage of fertile gametophores only at lower concentrations(10^–8-10^–6 mol dm^–3), and ferric citrate doesso at all levels. With salicylic acid, antheridia are induced3 d earlier. The number of gametophores as well as the percentageof fertile gametophores increases with the increase in coppersulphate concentration. Co-addition of EDTA (10^–5 moldm^–3) and copper sulphate inhibits both the responsesat higher levels. Among the chelating agents tried, Fe-EDTAis most effective in enhancing antheridial production. Key words: EDDHA, EDTA, Salicylic acid     

The Distribution of Nitrate Assimilation Between Root and Shoot in Vicia faba L.

Nitrate assimilation was examined in two cultivars (Banner Winterand Herz Freya) of Vicia faba L. supplied with a range of nitrateconcentrations. The distribution between root and shoot wasassessed. The cultivars showed responses to increased applied nitrateconcentration. Total plant dry weight and carbon content remainedconstant while shoot: root dry weight ratio, total plant nitrogen,total plant leaf area and specific leaf area (SLA) all increased.The proportion of total plant nitrate and nitrate reductase(NR) activity found in the shoot of both cultivars increasedwith applied nitrate concentrations as did NO₃^–: Kjeldahl-Nratios of xylem sap. The cultivars differed in that a greaterproportion of total plant NR activity occurred in the shootof cv. Herz Freya at all applied nitrate concentrations, andits xylem sap NO₃^–: Kjeldahl-N ratio and SLA were consistentlygreater. It is concluded that the distribution of nitrate assimilationbetween root and shoot of V. faba varies both with cultivarand with external nitrate concentration. Vicia faba L., field bean, nitrate assimilation, nitrate reductase, xylem sap analysis     

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.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta II. Aspects of Photoregulative Reaction

The photoregulation of carotenogenesis in Rhodotorula minutawas found to consist of tow phases, a temperature-independentphotochemical reaction (light process) and temperature-dependentbut light-independent biochemical reactions (dark process).These processes were separately examined by regulating the temperatureand were characterized as follows: 1) The quantity of carotenoid produced [C (µg g^–1)]and the rate of carotenoid production [Vc (µg g^–1hr^–1)] in the dark process were regulated by the lightdose [D (erg cm^–2)] to which cells were exposed in thelight process. These relationships were expressed by the equations:C=9.1 log D–62.0 and Vc=0.81 log D–5.60. This photoresponsefollowed the Roscoe-Bunsen reciprocity law. 2) The induced state toward carotenogenesis, once acquired inthe light process, was very stable, suggesting that the proposedphotochemical product is stable as an inducer of carotenogenesisand decreases only in conjunction with carotenoid biosynthesis. 3) The photochemical reaction was oxygen-independent, but subsequentdark reactions were completely dependent on oxygen. 4) Postulated compounds related to the photochemical reactionwere not metabolized in vivo. (Received September 12, 1981; Accepted February 20, 1982)     

Nitrate reductase from Dunaliella tertiolecta, purification and properties

The purification and properties of a nitrate reductase fromthe green alga Dunaliella tertiolecta are described. The enzymeis soluble, with a molecular weight greater than 500,000 andhas Km values of 0.26, 0.18, 0.10 and 0.06 m for NO₃^–,NADH, NADPH and FADH₂ respectively. Even at the highest specificactivity obtained, (0.86 µmoles NO₃^– reduced min^–1mg protein^–1) the enzyme retains the capacity to acceptelectrons from both NADH and NADPH. Unlike other nitrate reductasesit does not appear to be able to use reduced viologens as electrondonors. Its other properties are consistent with its being amolybdoflavoprotein of high molecular weight, which is alsoable to function as a cytochrome C reductase. ¹ Supported in part by the National Research Council of Canada. (Received June 18, 1972; )     

Photosynthesis in Panicum milioides, a Species with Reduced Photorespiration

The capacity for C₄ photosynthesis in Panicum milioides, a specieshaving reduced levels of photorespiration, was investigatedby examining the activity of certain key enzymes of the C₄ pathwayand by pulse-chase experiments with ¹⁴CO₂. The ATP$P₁ dependentactivity of pyruvate,P₁ dikinase in the species was extremelylow (0.14–0.18 µmol mg chlorophyll^–1 min^–1).Low activity of the enzyme was also found in Panicum decipiensand Panicum hians (related species with reduced photorespiration)and in Panicum laxum (a C₃ species). The antibody to pyruvate,P₁dikinase caused about 70% inhibition of the ATP$P₁ dependentactivity of the enzyme in P. milioides. The activity of NAD-malicenzyme and NADP-malic enzyme in ^P. ^milioides was equally low(approximately 0.1–0.2 µmol mg chlorophyll^–1min^–1) and similar to the activity in P. decipiens, P.hians and P. laxum. Photosynthetic pulse-chase experiments underatmospheric conditions showed a typical C₃-like pattern of carbonassimilation including the labelling of glycine and serine asexpected during photorespiration. During the pulse with ¹⁴CO₂only about 1% of the labelled products appeared in malate and2–3% in aspartate. During a chase in atmospheric levelsof CO₂ for up to 6 min there was a slight increase in labellingin the C₄ acids. The amount of label in carbon 4 of aspartatedid not change during the chase, indicating little or no turnoverof the C₄ acid via decarboxylation. The results indicate thatunder atmospheric conditions P. milioides assimilates carbondirectly through the C₃ pathway. Photorespiration as indicatedby the CO₂ compensation point may be repressed in the speciesby a more efficient recycling of photorespired CO₂. (Received June 8, 1982; Accepted July 22, 1982)     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

Biological activity of phyllosinol, a phytotoxic compound isolated from a culture filtrate of Phyllosticta sp.

1. Phyllosinol is a phytotoxic metabolite of Phyllosticta sp. Thissubstance at 100 µg/ml produced dark grey necrotic lesionson the leaf of red clover. Sensitivities of various plant speciesto phyllosinol differed both quantitatively and qualitatively.
2. Phyllosinol reduced root growth in rice seedlings by 60% at10^–4 M, whereas stimulation of root elongation occurredat a concentration range from 10^–9 to 10^–5 M.
3. Phyllosinolat 2.5x10^–4M promoted adventitious root formationin epicotylsof Azukia cuttings by about 100%. Promotion waspartly reducedby simultaneous application of cysteine.
4. IAA-induced elongationof isolated Avena coleoptile sectionswas inhibited by phyllosinolat a concentration range from 10^–5to 10^–3M.
5. Sulfhydrylcompounds, i.e. cysteine and glutathione relievedinhibitioncaused by phyllosinol in IAA-induced elongation ofAvena coleoptilesections.
6. GA₃-induced elongation of wheat leaf sections wasslightly inhibitedby phyllosinol at 10^–4M.
7. Phyllosinolalso has antibiotic activity. Among the organismstested, Phycomycetesand Gram-negative bacteria appeared mostsusceptible to phyllosinol.

(Received April 21, 1970; )