Comparison of production efficiency among field crops related to nitrogen nutrition and application

It has been generally considered that the low productivity of Leguminosae is caused by accumulation in the reproductive organs of a large amount of protein and lipid, since the biochemical costs of synthesizing these compounds is higher than that for carbohydrate. However, we report here on results which show that: the growth efficiencies (dry matter accumulated/ (dry matter accumulated + respiration)) of reproductive organs of Gramineae and Leguminosae were similar; the growth efficiency of rice in the vegetative stage was greater than that of soybean and field bean, regardless of nitrogen application rate; and when ¹⁴CO₂, ¹⁴C-sucrose or ¹⁴C-asparagine were introduced to the leaf at the maturation stage, respiratory loss of the introduced ¹⁴C was greater in soybean and field bean, especially in the light, than in rice. Thus, it is assumed that the low productivity in Leguminosae is caused by a larger respiratory loss under both dark and light condition in the shoot, and not in the reproductive organs.     

Measurements of N2 fixation by common bean in Central Brazil as affected by different reference crops

An experiment was conducted at EMBRAPA/CNPAF, Goiânia, Goias, Brazil, on a typic haplustox soil to evaluate growth and N₂ fixation-related parameters of Phaseolus vulgaris L. Bean lines, which had been selected for N₂ fixation at CNPAF, including production cultivars, germplasm bank entries, and parents and progenies of a cross made to improve this characteristic. Wheat (Triticum aestivum L.) and dwarf sorghum (Sorghum bicolor (L.) Moench) were evaluated as non-N₂-fixing reference crops for difference method (DM) and ¹⁵N isotope dilution technique (IDT) estimates of N₂ fixation. IDT estimates ranges from 4 to 18 kg N₂ fixed ha^-1. High variability associated with low levels of N₂ fixation precluded definitive identification of the best N₂ fixing bean lines. Due to differences in growth cycle and in patterns and amounts of soil N uptake during the season, neither of the reference crops tested appears to be an adequate control for either DM or IDT estimates of N₂ fixation. However, ranking of lines for effectiveness in N₂ fixation could be performed without the use of any reference crops.     

b-Type cytochromes in plasma membranes ofPhaseolus vulgaris hypocotyls,Arabidopsis thaliana leaves, andZea mays roots

Summary The plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb ₅₆₁ (cytb ₅₆₁), has its characteristic -band absorbance close to 561 nm wavelength at room temperature. Cytb ₅₆₁ was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb ₅₆₁ did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb ₅₆₁ was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb ₅₆₁ bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb ₅₆₁ from the plasma membranes of Arabidopsis leaves and maize roots showed similar Chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the Chromatographic behavior of cytb ₅₆₁ from different sources.Abbreviations cyt b ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - PAGE polyacrylamide gel electrophoresis     

Estimation of residual N effect of faba bean and pea on two succeeding cereals using15N methodology

A field experiment was conducted using¹⁵N methodology to study the effect of cultivation of faba bean (Vicia faba L.), pea (Pisum sativum L.) and barley (Hordeum vulgare L.) on the N status of soil and their residual N effect on two succeeding cereals (sorghum (Sorghum vulgare) followed by barley). Faba bean, pea and barley took up 29.6, 34.5 and 53.0 kg N ha^–1 from the soil, but returned to soil through roots only 11.3, 10.8 and 5.7 kg N ha^–1, respectively. Hence, removal of faba bean, pea and barley straw resulted in a N-balance of about –18, –24, and –47 kg ha^–1 respectively. A soil nitrogen conserving effect was observed following the cultivation of faba bean and pea compared to barley which was of the order of 23 and 18 kg N ha^–1, respectively. Cultivation of legumes resulted in a significantly higher A_N value of the soil compared to barley. However, the A_N of the soil following fallow was significantly higher than following legumes, implying that the cultivation of the legumes had depleted the soil less than barley but had not added to the soil N compared to the fallow. The beneficial effect of legume cropping also was reflected in the N yield and dry matter production of the succeeding crops. Cultivation of legumes led to a greater exploitation of soil N by the succeeding crops. Hence, appreciable yield increases observed in the succeeding crops following legumes compared to cereal were due to a N-conserving effect, carry-over of N from the legume residue and to greater uptake of soil N by the succeeding crops when previously cropped to legumes.     

Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

Trade-off relationships between some reproductive characteristics in plants with special reference to life history stragegy

Several reproductive triats in plants were studied in more than 200 populations of 61 wild species from diverse ecological conditions. As a result, it was found that there occur three distinct types of plants in the energy allocation patterns to reproductive structures (RA) and the propagule output per plant (P_N), i.e. (1) the number of propagules per plant increases in response to the increase in RA (Type I), (2) the number of propagules decreases in response to the increase in RA (Type II), and (3) the RA remains constant despite the great differences in the propagule number per plant. A conspicuous trade-off relationship was also discovered to occur between the RA to a single propagule (R_A) and the propagule output per plant (P_N), such that log R_A=logC−blot P_N, or R_A=C/P_N ^b =CP_N ^−b , where C is a constant. The three different ranges ofb-values were recognized, i.e.b<1.0,b>1.0, andb=1.0, which correspond to Type I, Type II, and Type III, respectively. Related problems to the concept ofr- andK-strategy are also discussed.     

Ontogenetic Changes of Photosynthetic and Dark Respiration Rates in Relation to Nitrogen Content in Individual Leaves of Field Crops

Ontogenetic changes of rates of photon-saturated photosynthesis (P _sat) and dark respiration (R _D) of individual leaves were examined in relation to nitrogen content (Nc) in rice, winter wheat, maize, soybean, field bean, tomato, potato, and beet. P _sat was positively correlated with Nc as follows: P _sat = CfNc + P _sat0, where Cf and P _sat0 are coefficients. The value of Cf was high in maize, medium in rice and soybean, and low in field bean, potato, tomato, and beet, of which difference was not explained by ribulose-1,5-bisphoshate carboxylase/oxygenase (RuBPCO) content. R _D was explained by P _sat and/or Nc, however, two models must be applied according to plant species. R _D related linearly with P _sat and Nc in maize, field bean, and potato as follows: R _D = a P _sat + b, or R _D = aNc + b, where a, a, b and b are coefficients. In other species, the R _D/P _sat ratio increased exponentially with the decrease of Nc as follows: R _D/P _sat = a exp(b Nc), where a and b are coefficients. Therefore, R _D in these crops was expressed as follows: In(R _D) = ln(a P _sat) + b Nc, indicating that R _D in these crops was regulated by both P _sat and Nc.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Nitrogen fixation in perennial forage legumes in the field

Nitrogen acquisition is one of the most important factors for plant production, and N contribution from biological N₂ fixation can reduce the need for industrial N fertilizers. Perennial forages are widespread in temperate and boreal areas, where much of the agriculture is based on livestock production. Due to the symbiosis with N₂-fixing rhizobia, perennial forage legumes have great potential to increase sustainability in such grassland farming systems. The present work is a summary of a large number of studies investigating N₂ fixation in three perennial forage legumes primarily relating to ungrazed northern temperate/boreal areas. Reported rates of N₂ fixation in above-ground plant tissues were in the range of up to 373 kg N ha^–1 year^–1 in red clover (Trifolium pratense L.), 545 kg N ha^–1 year^–1 in white clover (T. repens L.) and 350 kg N ha^–1 year^–1 in alfalfa (Medicago sativa L.). When grown in mixtures with grasses, these species took a large fraction of their nitrogen from N₂ fixation (average around 80%), regardless of management, dry matter yield and location. There was a large variation in N₂ fixation data and part of this variation was ascribed to differences in plant production between years. Studies with experiments at more than one site showed that also geographic location was an important source of variation. On the other hand, when all data were plotted against latitude, there was no simple correlation. Climatic conditions seem therefore to give as high N₂ fixation per ha and year in northern areas (around 60°N) as in areas with a milder climate (around 40°N). Analyzing whole plants or just above-ground plant parts influenced the estimate of N₂ fixation, and most reported values were underestimated since roots were not included. Despite large differences in environmental conditions, such as N fertilization and geographic location, N₂ fixation (Nfix; kg N per ha and year) was significantly (P<0.001) correlated to legume dry matter yield (DM; kg per ha and year). Very rough, but nevertheless valuable estimations of Nfix in legume/grass mixtures (roots not considered) are given by Nfix = 0.026DM + 7 for T. pratense, Nfix = 0.031DM + 24 for T. repens, and Nfix = 0.021DM + 17 for M. sativa.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

15N-determined dinitrogen fixation potential of genetically diverse bean lines (Phaseolus vulgaris L.)

Improvement of dinitrogen fixation in beans (Phaseolus vulgaris L.) will depend on the selection of superior plant genotypes and the presence of efficient rhizobial strains. This study was conducted to evaluate diverse bean lines for N₂ fixation potential using the¹⁵N-depleted dilution technique under field conditions in Wisconsin, USA. Plants of 21 bean lines and three non-nodulating isolines of soybean received appliin Wisconsin, USA. Plants of 21 bean lines and three non-nodulating isolines of soybean received applications of¹⁵N-depleted ammonium sulphate. Shoots harvested at the V₆, R₃ and R₇ stages and dry seeds were analyzed for total N using the Kjeldahl procedure, and the ratio of¹⁵N to¹⁴N was determind on a MAT 250 mass spectrometer. Nodule occupancy of the applied strain ofR. leguminosarum biovarphaseoli, CIAT 899, was determined in five of the bean lines. Total shoot N content showed a pattern of accumulation similar to shoot dry weight and fixed N₂ in the shoot. Based on shoot total N, N₂ fixed in the shoot and shoot dry weight Riz 30 and Preto Cariri were identified as being as good fixers as Puebla 152 and Cargamanto appear to begin N₂ fixation early. Furthermore, some bean lines that fixed considerable N₂ did not translocate a large amount of N to the grains. Preto Cariri accumulated 21.2 kg N ha⁻¹ in the seeds compared to Puebla 152 which accumulated 43.8 kg N ha⁻¹ of the fixed N₂ into the grains. At the early sampling, Puebla 152 and 22–27 had a considerable higher percentage of their crown nodules formed by the inoculant strain CIAT 899, than did Rio Tibagi which has been considered a poor N₂ fixer.     

Amount of nitrogen fixed by forage,pasture and grain legumes in Cyprus,estimated by the A-value and a modified difference method

The increasing need for protein at low cost has created a need to evaluate the biological nitrogen fixing potential of legumes in Cyprus. In field studies which were conducted over the growing years of 1982–3 and 1983–4, legumes which are traditionally grown in the country were evaluated for dry matter and nitrogen yield and biological nitrogen fixation (BNF). The legumes studied were medic (Medicago truncatula Gearth), ochrus vetch (Lathyrus ochrus L.), bitter vetch (Vicia ervilia L.) and faba bean (Vicia faba L. var major) in the first year and in addition chickpea (Cicer arietinum L.), woollypod vetch (Vicia dasycarpa Ten.) and tickbean (Vicia faba L. var minor) in the second year. Using the A-value method with barley and oats as reference crops, nitrogen (N) fixed by the various legumes in the first year was 30–50% and from 55–67% of total N yield for the two reference crops, respectively. In the second year the estimates of N fixed ranged from 70 to 80% with similar results obtained for the two reference crops barley and ryegrass. However, in the second year chickpea, which had limited nodulation, fixed only 40% of its N yield. Estimates of nitrogen from the atmosphere (Ndfa) obtained by the difference method (DM) were 10 to 14% lower than those from the A-value method. These results were obtained after correcting for the amount of N derived from the applied fertilizer. The two methods were highly correlated (r=0.98) for estimates of amount of BNF. The rates of N₂ fixation of uninoculated legumes which are nodulated by the indigenous populations of Rhizobium in Cyprus are comparable to those of legumes inoculated with selected strains of Rhizobium in other countries. An exception was the amount of N fixed by chickpea. The appearance of the first nodules at late stages of growth may be the reason for the low BNF of this crop.     

Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

Wounding and pathogen infection induce a chloroplast-targeted lipoxygenase in the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Chloroplastic LOXs are implicated in the biosynthesis of oxylipins like jasmonic acid and C₆ volatiles among others. In this study, we isolated the cDNA of a novel chloroplast-targeted Phaseolus vulgaris LOX, (PvLOX6). This gene is highly induced after wounding, non-host pathogen infection, and by signaling molecules as H₂O₂, SA, ethylene and MeJA. The phylogenetic analysis of PvLOX6 showed that it is closely related to chloroplast-targeted LOX from potato (H1) and tomato (TomLOXC); both of them are implicated in the biosynthesis of C₆ volatiles. Induction of PvLOX6 mRNA by wounding ethylene and jasmonic acid on the one side, and non-host pathogen, salicylic acid on the other indicates that common bean uses the same LOX to synthesize oxylipins in response to different stresses. PvLOX6 accession number: EF196866.     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

A proposed pathway of proton translocation through thebc complexes of mitochondria and chloroplasts

The cytochromebc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50–70% with minimal effect on electron transfer in the cytochromebc ₁ and cytochromebf complexes reconstituted into liposomes. Subsequent binding studies with cytochromebc ₁ and cytochromebf complexes indicate that DCCD specifically binds to the subunitb and subunitb ₆, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochromeb and aspartate 155 or glutamate 166 of spinach cytochromeb ₆. Moreover, similar inhibition of proton translocating activity and binding to cytochromeb and cytochromeb ₆ were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochromeb and cytochromeb ₆ is localized near the surface of the membrane but shielded from the external medium.     

Field observations on nitrogen catch crops. III. Transfer of nitrogen to the succeeding main crop

In temperate climates with a precipitation surplus during autumn and winter, nitrogen (N) catch crops can help to reduce nitrogen losses from cropping systems by absorbing nitrogen from the soil and transfer it to a following main crop. In two field experiments the catch crop species winter rye (Secale cereale) and forage rape (Brassica napus ssp. oleifera (Metzg.) Sinsk) or oil radish (Raphanus sativus spp. oleiferus (DC.) Metzg.) were planted end of August and 3 weeks later with a non-limiting supply of N and zero-N controls. In the next spring catch crops were incorporated into the soil. In Expt 1, N transfer was measured as (i) the N uptake of a potato test crop, grown with zero and 12.5 g m^–2 N applied, and (ii) the increase in soil mineral N (0–30 cm) in uncropped soil covered with polythene film. In Expt 2, N transfer was measured as the increase in soil mineral N in covered cylinders placed in uncropped soil (in situ incubation). Subsidiary laboratory incubations were performed in Expt 2. In Expt 1, the apparent recovery in potato of fertilizer N (R _f) was 0.56. The recovery in potato of N mineralized from 'native' N pools other than catch crop material (R _n) ranged from 0.43 to 0.51, depending on the value assumed for the depth of N extraction by potato roots. The average recovery in potato of incorporated catch crop N (R _c) was 0.34. Expressed as `fertilizer N replacement factor' (F _r) the latter was 0.61 (i.e. 1 kg of N in catch crop material counts for 0.61 kg fertilizer N). Under the film in Expt 1 the fraction net mineralization of incorporated catch crop N (M _n) was 0.36 on August 11 and 0.43 on October 18. In Expt 2, the average value of M _n was 0.31, which was lower than in Expt 1 and probably associated with the drier soil in Expt 2. In the laboratory incubations (20°C) M _n showed values up to 0.54 after 84 days with the largest rates of change in mineralization occuring early after the start of the incubation. In conjunction with literature data it is concluded that cultivation of nitrogen catch crops shows promise as a means to reduce N input and N losses in temperate climates with wet winters.