Carbon monoxide sensitivity of cytochrome oxidase in wheat leaves in relation to dwarfing genes (Rht) in wheat cultivars

Summary Leaves of young seedlings of a number of tall cultivars of wheat, lacking the dwarfing Rht genes, readily responded to a brief 2 min exposure to CO, as assessed by in vivo aerobic assay of nitrate reductase. This test depends on the inhibition of cytochrome c oxidase by CO, which in turn renders cytosolic NADH available for the reduction of nitrate to nitrite in vivo. Semi-dwarf cultivars of wheat (Rht present) did not respond to CO in this way. Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃, during in situ respiration of leaves from tall and semi-dwarf plants which are likely to be under genetic control.     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

Light and ethylenediamine tetraacetic acid mediated differential effects of ammonium on nitrate reductase activity in maize seedlings

Abstract Effect of ammonium on in vivo activity of nitrate reductase in roots, shoots and leaves of maize (Zea mays L.) seedlings was studied in relation to light/dark conditions and EDTA supply. Supply of 5 mM (NH₄)₂SO₄ increased the steady state level of enzyme only in leaves and in light, while it had no effect in roots and shoots and in the dark. The substrate induction of enzyme was also little affected by 1 to 10 mM (NH₄)₂SO₄ in roots and shoots. In the leaves the activity in the dark was either inhibited (minus EDTA) or stimulated (plus EDTA) by 5 to 10 mM (NH₄)₂SO₄. The activity was stimulated in the light also in the presence of EDTA at higher concentrations of ammonium. When different concentrations of ammonium were supplied without any exogenous nitrate in the light, the enzyme activity increased at low concentration and was either inhibited or unaffected at higher concentrations depending upon the tissue used. Supply of EDTA with ammonium modified its effect to some extent. It is suggested that the effect of ammonium on nitrate reductase activity depends upon the tissue used and the effective concentration of the ammonium.     

Differential carbon monoxide sensitivity of cytochrome C oxidase in the leaves of c(3) and c(4) plants

CO sensitivity of cytochrome a₃ in the leaves of a number of C₃ and C₄ plants was monitored by the nitrate reductase assay under differing CO to O₂ ratios. All the C₃ plants were relatively insensitive to CO and required a high CO to O₂ ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to O₂ ratios of 10 or less. On the other hand, the leaves of all C₄ plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to O₂ ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from mesophyll or bundle sheath cells or both, lacked tight respiratory control.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

The Systems I, II and III in the Photosynthetic Cycle of Electron Transfer

• 1 Two separate light reactions may be distinguished: (I) the reduction of ferredoxin and NADP probably by oxidation of carotene to xanthophyll; (II) the oxida tion of cytochrome f by chlorophyll (probably a). Reaction II implies a return of electrons to the pigments, system III, thus maintaining its normal steady state of oxidation-reduction. The xanthophyll is hereby again reduced to carotene.
• 2 System I is sensitive to violet-blue-green and probably also infrared light. Carotene absorbs in these regions, ferredoxin in blue-violet. System II is primarily sensitive to red light but also to blue-violet. Both chlorophyll and cytochromes absorb in the latter region, the cytochromes also in green.
• 3 The response of systems I and II to different spectral regions was studied by means of a special spectrophotometric flash technique, enabling precise measure ments of the band-height of the enzymes involved. The initial photic reactions of systems I and II, viz., the reduction of ferredoxin-NADP and the oxidation of cyto chrome f show a full turnover in less than 0.1 ms but the transfer between systems I and II by means of which the cytochromes are reduced is slowed down to about 10^?1-10^?2 s. The initial effect may thus be observed during ca. 0.1 s. At continuous illumination the displacement of the steady states of the enzymes may last up to several seconds and then return to a state of only partial reduction. Erroneous inter pretations of these phenomena are corrected.
• 4 In the blue-sensitive system I ferredoxin alone mediates the reduction of NADP but the possibility of the presence of other factors capable of dark chemical elec tron transfer is discussed. In the red-sensitive system II three cytochromes operate, viz., f, b₃ and b₆. Spectrophotometric evidence for the existence of two cytochromes b is presented. Cytochromes b₆ and f are approximately synchronously oxidized and reduced, whereas b₃ reacts somewhat independently. Cytochrome b₃ probably acts as a decharger of OH^? and compensates for the capture of H⁺+ e^? at the reduction of triphosphopyridine nucleotide (NAD) or of electrons by other oxidants.
• 5 The transfer of electrons between systems I and II maintains a reversible steady state of oxidation-reduction that may be moved to one side or the other not only by monochromatic light but also in the dark under influence of N₂, O₂, the ratios NADP/NADPH and ADP/ATP, and various added substances. Spectrophotometric measurements in UV show that a flavoprotein participates in the multiple steady state.
• 6 The investigations illustrate many intricate technical problems that are too frequently overlooked. Photostructural reactions must be eliminated by referring band-heights to an isosbestic level. The photosynthetic activity is strongly dependent on light-scattering. Reliable measurements of cytochromes must be made in the α-region owing to a strong interference of rapid changes of the ratio carotene/xanthophyll in the region of the γ-bands.

     

The invisible copper of cytochrome c oxidase. pH and ATP dependence of its midpoint potential and its role in the oxygen reaction.

Formation of the CO compound has been studied in intact mitochondria, submitochondrial particles and isolated cytochrome oxidase. The reaction requires the prior reduction of both cytochrome a₃ and one other single-electron acceptor. It is inferred that the second acceptor is the “invisible” copper which is undetectable by both optical and spin resonance spectroscopy. The overall process can be viewed as two single electron steps plus a ligand binding reaction. At high concentrations of CO, when titrations are performed at oxidation-reduction potentials significantly above the midpoints of either cytochrome a₃ or “invisible” copper, appearance of the CO compound follows a strict n = 2 (2-electron) relationship. Its midpoint potential is also dependent on the prevailing concentration of CO and is increased by approx. 30 mV for each tenfold increase in the level of CO. At redox potentials approaching the midpoints of cytochrome a₃ or “invisible” copper, significant deviations from n = 2 behavior are apparent which are readily detectable experimentally using low CO concentrations.A mathematical analysis of this model is presented and the oxidation-reduction properties of the CO compound are utilized to determine the midpoint potential of the “invisible” copper. This value is estimated to be 340 ± 10 mV at pH 7.8, independent of pH and the prevailing $\text{sol}\text{[ATP]}\text{[ADP]}\text{× [}\text{P}{}_1\text{]}$ ratio.By analogy with the observations on CO binding, the primary intermediate in the oxidase reaction with oxygen is concluded to be a bridged a₃²⁺-O₂-Cu¹⁺ complex. The initial reduction of molecular oxygen can then proceed via a thermodynamically favorable two-electron step to form a bridged peroxide intermediate. Subsequent reduction to water may later occur by way of two single-electron steps or one two-electron step.     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c₁ and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c₁a and a₃ were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a₃ were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.     

Cytochrome oxidase sensitivity to carbon monoxide in leaves of various tall and dwarf wheat cultivars and their crosses

In order to investigate the possible role of Rht genes in the regulation of the redox condition of cytochrome a₃ (cytochrome c oxidase) during steady-state respiration, wheat cultivars belonging to one of two groups - NP 710, NP 846 and NP 875 belonging to the tall group and Olesons dwarf, HD 1982 and HD 2122 of the dwarf group - and the reciprocal crosses between the varieties of these two groups were examined for carbon monoxide (CO) sensitivity in terms of the inhibition of mitochondrial electron transport. Leaves of young wheat seedlings were used. Differences in the redox state of cytochrome a₃ were monitored using the in vivo aerobic assay of nitrate reduction after a 1-min exposure to CO. Dwarf cultivars possessing Rht genes responded marginally (᜖%) to CO inhibition, whereas the response of tall cultivars to CO was higher (51-70%). Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃ during in situ respiration of leaves from tall and dwarf plants that are likely to be controlled by cytoplasmic factors.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

A computer-operated routine of gas exchange and optical measurements to diagnose photosynthetic apparatus in leaves

Photosynthesis is a complex process whose rate is affected by many biochemical and biophysical factors. Fortunately, it is possible to determine, or at least estimate, many of the most important parameters using a combination of optical methods and gas transient analyses. We describe here a computer‐operated routine that has been developed to make detailed assessments of photosynthesis at a comprehensive level. The routine comprised the following measurements: steady‐state light and CO₂ response curves of net CO₂ assimilation at 21 and 2 kPa O₂; transients from limiting to different saturating CO₂ concentrations at 2 kPa O₂; post‐illumination CO₂ fixation transient; dark–light induction of O₂ evolution; O₂ yield from one saturating single‐turnover flash; chlorophyll fluorescence F₀, F_s and F_m during the light and CO₂ response curves; leaf transmission at 820 nm (P700⁺) during the light and CO₂ response curves; post‐illumination re‐reduction time of P700⁺. The routine was executed on a two‐channel fast‐response gas exchange measurement system (A. Laisk and V. Oja: Dynamic Gas Exchange of Leaf Photosynthesis. CSIRO, Canberra, Australia). Thirty‐six intrinsic characteristics of the photosynthetic machinery were derived, including quantum yield of CO₂ fixation (Y_CO2), time constant of P700 re‐reduction (τ′), relative optical cross‐sections of PSII and PSI antennae (a_II, a_I), PSII and PSI density per leaf area unit, plastoquinone pool, total mesophyll resistance, mesophyll diffusion resistance, V_m, K_m(CO₂) and CO₂/O₂ specificity of Rubisco, RuBP pool at CO₂ limitation (assimilatory charge). An example of the routine and calculations are shown for one leaf and data are presented for leaves of 8‐year‐old‐trees of two birch clones growing in Suonenjoki Forest Research Station, Finland, during summer 2000. Parameters Y_CO2, basic τ′, a_II, a_I, K_m(CO₂) and K_s varied little in different leaves [relative standard deviation (RSD) < 7%], other parameters scattered widely (RSD typically 10–40%). It is concluded that the little scattered parameters are determined by basic physico‐chemical properties of the photosynthetic machinery whereas the widely scattered parameters are adjusting to growth conditions. The proposed non‐destructive routine is suitable for diagnosing the photosynthetic machinery of leaves and may be applied in plant ecophysiology and in genetic engineering of plants.     

Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b₅ or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.

Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.

     

The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions

The assimilation of nitrate under dark-N₂ and dark-O₂ conditions in Zea mays leaf tissue was investigated using colourimetric and ¹⁵N techniques for the determination of organic and inorganic nitrogen. Studies using ¹⁵N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N₂ conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O₂ was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.     

Light-dependent fluorescence variations in intact leaves

Transient variations in the fluorescence from intact Phytolaccaamericana leaves after the onset of illumination were measuredunder various light and dark conditions. Dark-adapted leaveswhen illuminated with strong light underwent an intensity variationwith a peak; the fluorescence intensity reaching its peak severalseconds after the onset of illumination then decreasing to asteady level. The peak height relative to the steady level increasedwith the increasing intensity of actinic light. Pre-illuminationof the dark-adapted leaves with strong light caused a markedlowering of the peak. About 20 min of dark incubation was requiredfor the light-adapted leaves to return to the dark-adapted state.All of the action spectra, for the peak, the steady level andthe effect of light in post-illumination to inhibit recoveryto the dark state, showed high bands due to chlorophyll b andcarotenoid absorption and low bands due to chlorophyll a absorption.We concluded that the light absorbed by photosystem 2 is responsiblefor these phenomena. (Received April 21, 1975; )     

Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandrium tamarense (Gonyaulacales, Dinophyceae)

The growth and photosynthesis of Alexandrium tamarense (Lebour) Balech in different nutrient conditions were investigated. Low nitrate level (0.0882 mmol/L) resulted in the highest average growth rate from day 0 to day 10 (4.58 × 10² cells mL^?1 d^?1), but the lowest cell yield (5420 cells mL^?1) in three nitrate level cultures. High nitrate‐grown cells showed lower levels of chlorophyll a‐specific and cell‐specific light‐saturated photosynthetic rate (P_m^{chl a} and P_m^cell), dark respiration rate (R_d^chla and R_d^cell) and chlorophyll a‐specific apparent photosynthetic efficiency (α^chla) than was seen for low nitrate‐grown cells; whereas the cells became light saturated at higher irradiance at low nitrate condition. When cultures at low nitrate were supplemented with nitrate at 0.7938 mmol/L in late exponential growth phase, or with nitrate at 0.7938 mmol/L and phosphate at 0.072 mmol/L in stationary growth phase, the cell yield was drastically enhanced, a 7–9 times increase compared with non‐supplemented control culture, achieving 43 540 cells mL^?1 and 52 300 cells mL^?1, respectively; however, supplementation with nitrate in the stationary growth phase or with nitrate and phosphate in the late exponential growth phase increased the cell yield by no more than 2 times. The results suggested that continuous low level of nitrate with sufficient supply of phosphate may facilitate the growth of A. tamarense.     

Interaction of Ca2+ and H+ with heme A in cytochrome oxidase

Ca²⁺ ions shift the absorption spectrum of reduced cytochromea in mitochondria by acting from the outside of the membrane. In isolated cytochrome oxidase the shift may be induced by either Ca²⁺ or H⁺, the apparent pK varying between 6.20 and 5.75 depending on the state of cytochromea ₃. Studies of the Soret band show that Ca²⁺ also shifts the spectrum of ferrocytochromea ₃ in isolated oxidase in contrast to the situation in mitochondria or isolated oxidase reconstituted into liposomes. Model studies with reduced bis-imidazole heme A reveals an analogous spectral shift induced by Ca²⁺. Esterification of the propionate carboxyls of heme A abolishes the spectral shift, suggesting that it is due to interaction of Ca²⁺ with these groups. When taken together with the data with intact mitochondria, this suggests that the propionate side chains of cytochromea are accessible to Ca²⁺ and H⁺ from the outside of the mitochondrial membrane. In the soluble enzyme both hemesa anda ₃ are accessible. Thus hemea may be located near the outside of the inner membrane whereas hemea ₃ experiences a different environment in which no Ca²⁺ shift occurs.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

The nitrogen balance of Raphanus sativus X raphanistrum plants. I. Daily nitrogen use under high nitrate supply

Abstract Growth-chamber cultivated Raphanus plants accumulate nitrate during their vegetative growth. After 25 days of growth at a constant supply to the roots of 1 mol m^?3 (NO^?₃) in a balanced nutrient solution, the oldest leaves (eight-leaf stage) accumulated 2.5% NO^?₃-nitrogen (NO₃-N) in their lamina, and almost 5% NO₃-N in their petioles on a dry weight basis. This is equivalent to approximately 190 and 400 mol^?3 m^?3 concentration of NO^?₃ in the lamina and the petiole, respectively, as calculated on a total tissue water content basis. Measurements were made of root NO^?₃ uptake, NO^?₃ fluxes in the xylem, nitrate uptake by the mesophyll cells, and nitrate reduction as measured by an in vivo test. NO^?₃ uptake by roots and mesophyll cells was greater in the light than in the dark. The NO^?₃ concentration in the xylem fluid was constant with leaf age, but showed a distinct daily variation as a result of the independent fluxes of root uptake, transpiration and mesophyll uptake. NO^?₃ was reduced in the leaf at a higher rate in the light than in the dark. The reduction was inhibited at the high concentrations calculated to exist in the mesophyll vacuoles, but reduction continued at a low rate, even when there was no supply from the incubation medium. Sixty-four per cent of the NO^?₃ influx was turned into organic nitrogen, with the remaining NO^?₃ accumulating in both the light and the dark.