PHOTOSYNTHETIC NITRITE REDUCTION BY DITHIOERYTHRITOL AND THE EFFECT OF NITRITE ON ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS

Abstract. Photosynthetic reduction of nitrite to ammonia with type C chloroplasts from the heterocont alga Bumilleriopsis filiformis was investigated using 3,6-diaminodurene/ascorbate and 3,6-diaminodurene/dithioerythritol (DAD/DTE) as electron donor couple. Rates approach 6–10 μmol NO^-₂ reduced/mg chlorophyll/h and are steady for up to 30 min. The presence of oxygen or NADP⁺ only slightly diminished the rates of nitrite reduction obtained with DAD/DTE. Illuminated chloroplasts reduce oxygen in the presence of DAD/DTE at 135 μmol/mg chlorophyll/h without acceptor supplied. Photosynthetic oxygen uptake by this system in the presence of ferredoxin and NO^-₂, however, is inhibited to 42% by nitrite reductase with concurrent nitrite reduction. NO^-₃ and NO^-₂ have no effect on photosystem I-mediated NADP⁺ reduction, NO^-₂ (10 m M ) inhibits ferricyanide-mediated oxygen evolution to 72%. Also photosystem II reactions assayed e.g. with silicomolybdate are inhibited significantly by NO^-₂ (1 m M ), but only slightly by NO^-₃. Nitrite reductase is inhibited by p -chloromercuribenzoate ( p CMB), and this inhibition is prevented by DTE. Results suggest that photosynthetic nitrite reduction can cope with low concentrations of either compound, provided relevant thiol groups are protected.     

KINETIC ANALYSIS OF THE FLUORESCENCE INDUCTION IN 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA POISONED CHLOROPLASTS

Abstract— The size of the area over the fluorescence rise curve of chloroplasts is a measure of the total number of quanta utilized in photosystem II during the fluorescence induction, while the growth of the area reflects the progress of photochemical events. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the growth kinetics of the area are affected by the reoxidation of the primary acceptor Q ^- with stored oxidizing charges on the donor side of system II.
At low light intensities, a slow component of this back reaction may limit the steady state fluorescence emission. At higher intensities, however, the fluorescence rise is limited solely by photochemical events, although fast thermochemical reactions like the immediate recombination of photochemically separated charges may affect the efficiency of the photochemistry.
A kinetic analysis of the area growth at moderate light intensities revealed that it occurred in two first order phases which were described by the rate constants k _α and k _β. The biphasic nature suggested a sequential two-electron reduction of the primary acceptor Q , or the presence of two different types of photochemical centers in system II. The rate constants were light intensity dependent. They also were affected by changes in pH, by an addition of NH₂OH, or by a preillumination with short flashes prior to addition of DCMU. It is suggested that the pH of the medium, the presence of NH₂OH, and the flash induced state S_n of the water splitting enzyme, control the values of k _α and k _β by changing the rate constants of electron carrier interactions in the reaction center complex, with a resulting modification of the frequency of back reactions between the primary donor and the primary acceptor.     

DIURNAL VARIATIONS IN LUMINESCENCE IN Scenedesmus obtusiusculus (CHLOROPHYCEAE) INDUCED BY 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA

Abstract— Luminescence from synchronously cultured Scenedesmus obtusiusculus cells was measured with a high sensitivity photon counter. Recording of light emission was initiated 0.2 s after switching off actinic light. Luminescence decay was separated into two phases: one for decay to 10⁴ pulses s^-1, the other for decay from 10⁴ to 10³ pulses s^-1. Most photons are emitted during the rapid decay to 10⁴ pulses s^-l. Only small diurnal variations of the two phases could be observed in controls. Treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased both the total number of photons emitted and the time required to reach the 10⁴ pulses s^-1 level. No diurnal rhythmicity was induced by DCMU in the first phase but DCMU induced a pronounced diurnal variation in decay time in the second phase of luminescence parallelled by a periodicity in the number of photons emitted. The results indicate that DCMU interferes with the participation of PS I in luminescence. The chlorophyll alb ratio was constant during the life cycle of the cells. No relation could be observed between luminescence and the diurnal rhythmicity in photosynthesis that is characteristic for synchronized unicellular algae.     

OXYGEN QUENCHING OF CHLOROPHYLL FLUORESCENCE IN CHLOROPLASTS

Abstract— Three phases of chlorophyll a fluorescence quenching by O₂ are observed in green plants. The effects of various inhibitors on photosynthetic partial processes in chloroplasts were investigated in attempts to (1) localize the O₂-quenching sites and (2) assess possible physiological significance of O₂-quenching. Our results localize the most sensitive (and presumably functionally important) phase to a site between plastoquinone and the photosystem I acceptor, chlorophyll (P₇₀₀), possibly plastocyanin. It is suggested that PC may transfer electrons to oxygen in addition to P₇₀₀.     

USE OF THE FLUORESCENT LANTHANIDE Tb3+ AS A PROBE FOR CATION-BINDING SITES ASSOCIATED WITH ISOLATED CHLOROPLAST THYLAKOID MEMBRANES

Abstract— The fluorescent properties of the rare-earth ion, Tb³⁺ have been utilized to probe the nature of cation-binding sites associated with thylakoid membranes. At low concentrations (< 100μ M ), Tb³⁺ was observed to inhibit the increase in chlorophyll a fluorescence normally seen on adding 5 m M MgCl₂ (or 100 m M NaCl) to isolated, broken chloroplasts. We also observed under these conditions, the appearance of a new band around 280 nm in the excitation spectrum of Tb³⁺ ion fluorescence. However, similar changes in Tb³⁺ fluorescence could be observed in the presence of a membrane-free preparation of chloroplast coupling factor protein (CF₁). From this and other results it is concluded that changes in Tb³⁺ fluorescence reflect an association of the ion with CF₁ followed by intermolecular transfer of excitation energy from protein ligands (possibly un-ionized tyrosine residues) to the lanthanide. The interaction of Tb³⁺ with sites which control chlorophyll a fluorescence does not seem to modify Tb³⁺ fluorescence, suggesting that in this case, simple membrane-bound ligands such as carboxyl or phosphate groups are involved.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

THE SUPEROXIDE ANION AS ELECTRON DONOR TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN

Abstract— In isolated respiratory multienzyme complexes of beef heart mitochondria the b -type cytochromes can be photoreduced in presence of flavin via the superoxide anion. O^-₂ does not reduce cytochrome c ₁. In an anaerobic system, FMNH₂ formed by irradiation with blue light in presence of EDTA reduces cytochromes b and c ₁ The possible implication of O^-₂ in the electron transfer from flavin/flavoprotein to cytochrome b in blue light-controlled biological processes is discussed.     

Changes of Chlorophyll(ide) Fluorescence Yield Induced by a Short Light Pulse as a Probe to Monitor the Early Steps of Etioplast Phototransformation in Dark-Grown Leaves

The fluorescence yield of chlorophyll(ide) (Chl[ide]) excited by weak modulated light was recorded at room temperature during a 2 h period after a short actinic light pulse that transformed all photoactive protochlorophyllide in dark-grown barley leaves. A typical pattern of fluorescence yield variations was found whatever the age of the leaf but with age-dependent changes in rates. Its successive phases were related to the Chl(ide) spectral shifts observed in low-temperature emission spectra. The fluorescence yield started at a high level and strongly declined during the formation of Chlide₆₉₅ from Chlide₆₆₈ within a few seconds. It increased to a transient maximum during the Shibata shift (15–25 min) that resulted in Chl(ide)₆₈₂. A final, slow decrease to a steady state occurred during the final red shift to Chl₆₈₅. Pretreatments with δ-aminolevulinic acid, chloramphenicol or 1, 10-phenanthroline resulted in correlated modifications of Chl(ide) fluorescence yield transients and shifts of the low-temperature Chl(ide) emission band. The complex response of the final decrease phase of the fluorescence yield to these compounds suggests that it results both from the assembly of photosynthetic Chl proteins and from the reorganization of the etioplast membrane system. From these results it is concluded that continuous recordings of Chl(ide) fluorescence yield after a short light pulse represent a useful tool to monitor the kinetics of pigment–protein organization and primary thylakoid assembly triggered by Pchlide photoreduction.     

A STUDY OF THE FLASH INDUCED 518 NM ABSORBANCE CHANGE KINETICS UNDER ANAEROBIC CONDITIONS

The preillumination induced acceleration of the flash-induced 518 nm absorbance change (ΔA₅₁₈) decay was studied in lettuce leaves and chloroplasts. In leaves, the acceleration was inhibited by DCMU or reversibly by removal of oxygen. In chloroplasts with added ADP and phosphate and/or reconstructed electron transport, the acceleration was also inhibited by DCMU or the lack of O₂.
Anaerobic inhibition of ΔA₅₁₈ decay acceleration was no longer observed when hydroxylamine replaced water as electron donor to PSII. Anaerobiosis was also shown to reversibly inhibit the initial rate of FeCN reduction in chloroplasts. These results suggest the mechanism of anaerobic inhibition of ΔA₅₁₈ decay acceleration to be associated with the O₂ evolving system.     

CONCENTRATION-DEPENDENT LIFETIMES OF Cu(NN)+2 SYSTEMS: EXCIPLEX QUENCHING FROM THE ION PAIR STATE

Abstract— The concentration dependence of the lifetimes of the charge transfer excited states of Cu(dmp)⁺₂ and Cu(dpp) ⁺₂ has been investigated in CH₂C1₂ solution at 20^°C. (dmp denotes 2,9-dimethyf-1,10-phenanthroline, and dpp denotes 2,9-diphenyl-l,10-phenanthroline.) In dilute solution (< 30 μM) the lifetime of Cu(dmp)⁺₂, is 95 ± 5 ns, independent of the anion. At higher concentrations the lifetime decreases, in most cases, to a limiting value that depends upon the counterion. The measured limiting lifetimes range from 38 ± 3 ns for CIO^-₄ to 78 ± 5 ns for PF^-₆. The anion-induced quenching is attributed to exciplex quenching which is mediated by an ion pair which exists in the ground state. The results imply that the quenching ability of the anions follows the order BPh-₄ < PF -₆, < BF-₄ < CIO -₄ < NO-₃ which is consistent with previous estimates of donor strength. The lifetime of Cu(dpp)⁺₂ is also concentration dependent, but the effect is much smaller because the phenyl substituents impede attack by the anion.     

PHOTOREDUCTION OF NAD TO NADH IN SEMICONDUCTOR DISPERSIONS

Abstract— Band gap illumination of TiO₂ (anatase) dispersions in weakly alkaline solutions of nicotinamide-adenine-dinucleotide (NAD⁺) leads in the presence of rhodium trisbipyridyl complex [Rh(bipy)³⁺₃], to continuous generation of biologically active cofactor NADH. Effects of pH, NAD⁺ and Rh(bipy)³⁺₃ concentration on the efficiency of this photoconversion process are investigated. The reaction proceeds already in aqueous solution in the absence of external electron donors but it is enhanced significantly in the presence of 10% methanol.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

PHOTOCLEAVAGE OF DNA IN THE PRESENCE OF SYNTHETIC WATER-SOLUBLE PORPHYRINS

In the presence of oxygen and visible light, various synthetic water-soluble porphyrins cleave pBR 322 plasmid supercoiled DNA (form I) producing relaxed (form II) and linear (form III) DNA corresponding to single-strand and double-strand breaks respectively. Large variations are observed in the efficiency of the porphyrins containing a diamagnetic metal or no metal at all. Singlet oxygen (¹O₂) seems to be involved in the mechanism of cleavage consistent with the inhibitory effect of the azide anion, N^–₃. The higher efficiency of cationic porphyrins (as compared to anionic ones) is due to their greater affinity for DNA as shown by experiments carried out at either high ionic strength or in the presence of the surfactant, sodium dodecyl sulfate.     

DETERMINATION OF THE KINETICS OF THE BACK REACTION OF PHOTOSYSTEM II IN THE PRESENCE OF 3–(3,4-DICHLORPHENYL)-1,1-DIMETHYLUREA FROM LUMINESCENCE MEASUREMENTS

Abstract— An alternative method to that used by Mar and Roy (1974) for the determination of the kinetics of the back reaction of photosystem II from the luminescence decay curve in the presence of 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) has been suggested. The new theory relies upon two hypotheses: the well-known recombination hypothesis of luminescence and the assumption that the luminescence yield in the seconds region is given by the variable part of the live fluorescence yield. The second hypothesis was introduced since assuming a constant luminescence yield results in kinetic data that are not consistent with measurements of the kinetics of the back reaction by the restoration of the area over the fluorescence rise curve. The dependence of the live fluorescence yield from the concentration of closed PS II traps was assumed to be represented by Delosme's expression originally derived for the rise curve of the fluorescence yield in the presence of DCMU.
The theory is based on the fact that then the partial and total light sums of luminescence are simple functions of the concentration of the primary electron acceptor Q^- of PS II. Thus, after integrating the luminescence decay curve the theory permits a convenient evaluation of the kinetics of the back reaction [Q^-]( t ) in terms of the partial and total light sum.
This method was applied in order to determine the kinetics of the back reaction in Chlorella fusca in the presence of DCMU. It is shown that the kinetics of deactivation of the S, state can be described using the expression for the kinetics of the back reaction derived by Mar and Roy. As an alternative explanation, a biphasic first order decay of S₂ is proposed.     

FLUORESCENCE INDUCTION IN THE RED ALGA PORPHYRIDIUM CRUENTUM

Abstract— The intensity dependence and the spectral changes during the fast (sec) and the slow (min) transient of chlorophyll (Chl) a fluorescence yield, measured at 685 nm, have been analyzed in the red alga Porphyridium cruentum . Both the fast and the slow fluorescence yield changes are affected differently by the inhibitors of electron transport ( e.g ., DCMU) and by the uncouplers of phosphorylation (atebrin and FCCP). Fixation of Porphyridium cells with glutaraldehyde abolishes most of the fluorescence yield changes except for the so-called very fast ( OI ) phase. The same fixed cells, however, reduce DCPIP (a Hill oxidant) but do not evolve O₂ when CO₂ is used as electron acceptor. We interpret these and other results by the hypothesis that fluorescence transients in intact cells are linked to both electron transport and the energy dependent structural changes in the thylakoid membrane.     

FLUORESCENCE INDUCTION IN ALGAE AND CHLOROPLASTS

Abstract —The first phases of the fluorescence transient elicited by illumination of dark-adapted algae or isolated chloroplasts (biphasic rise φ_***v - φ_I - _P) have previously been shown to be controlled by two quenchers: Q , the primary acceptor of Photosystem 2 interacting with the secondary acceptor pool A ; and R , a non-photochemical quencher which goes into a non-quenching state as A is reduced.
The dependence of the kinetics of φ decay after illumination upon the redox state of A was studied. It is suggested that some of the centres are in a disconnected state, where electron transfer between Q and A is hindered, the amount of such centres being correlated to the reduction state of A . The implications of this hypothesis on the problem of the variation of the Q-A 'equilibrium constant' under different experimental conditions, and on Murata's 'weak light effect' are discussed.
The effect of 3-(3,4 dichlorophenyl)-1,1 dimethylurea (DCMU) on R is shown to depend upon the redox state of A . A DCMU-induced shift of the midpoint potential of R may account for this dependence.
Evidence is given suggesting that the transient reduction of A which occurs in algae during the φ_I-φ_P rise is controlled by an induction process on the acceptor side of Photosystem 1.     

INDEPENDENT EFFECTS OF PHYTOCHROME AND NITRATE ON NITRATE REDUCTASE AND NITRITE REDUCTASE ACTIVITIES IN MAIZE

Abstract— Involvement of phytochrome in the regulation of nitrate reductase (NR) and nitrite reductase (NIR) activities in excised, etiolated leaves of Zea mays (L.) variety 'Ganga-5' is demonstrated using low energy and high irradiance responses of phytochrome action. Photoreversibility by far-red light of red light stimulated increases in NR and NIR activities was lost by 2 h. Red light given to the leaves, when induction by NO^-₃, was saturated, further increased both enzyme activities. Even if red light was given 4–8 h before NO^-₃, it still increased both NR and NIR activities.     

QUENCHING OF THE EXCITED STATES OF 2-PHENYLBENZOXAZOLE BY AZIDE ANION. FLUORESCENCE AND ESR STUDY

Electron spin resonance, spin-trapping and fluorescence techniques demonstrate that 2-phenylbenzoxazole (P) participates in photo-induced reactions with alcohols and electron donors like the azide ion. Irradiation of Pat 300 nm in deaerated ethanol produces ethoxyl and hydroxyethyl radicals which can be detected with the spin trap, 5,5-dimethyl-l-pyrroline-l-oxide (DMPO). However, irradiation of P in the presence of N^-₃ leads to the appearance of the azide radical, N^-₃, which also reacts with DMPO. Studies with the nitroxyl radical, 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), suggest that electron transfer from the azide anion to an excited state of P yields the semi-reduced sensitizer, P^-, which in turn reacts with TEMPO. The effect of sodium azide upon the fluorescence intensity and lifetime of P in aqueous ethanol has also been studied.     

"ENERGY"-DEPENDENT QUENCHING OF CHLOROPHYLL FLUORESCENCE and THERMAL ENERGY DISSIPATION IN INTACT LEAVES DURING INDUCTION OF PHOTOSYNTHESIS

Abstract— Intact leaves, previously adapted to darkness for a prolonged period of time, were suddenly illuminated with a strong, photosynthetically saturating, white light (ca 1500 μmol m⁻² s^_1), resulting in the rapid establishment of a large energy-dependent chlorophyll fluorescence quenching (q_E) as shown by in vivo fluorescence measurements with a pulse amplitude modulation technique. Two different photothermal methods, photoacoustic spectroscopy and photothermal deflection spectroscopy, were used to monitor thermal deactivation of excited pigments during the dark-light transitions. The in vivo photothermal signals measured with both techniques were shown to remain constant during induction of photosynthesis under high light conditions, suggesting that, in contrast to current hypotheses, energy-dependent quenching q_E is not associated with significant changes in thermal dissipation of absorbed light energy in the chloroplasts. When photosynthesis was induced with a low-intensity modulated light, a noticeable decrease in the heat emission yield was observed resulting from the progressive activation of the competing photochemical processes.