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.     

LASER PHOTOLYSIS OF 3-METHYLLUMIFLAVIN

Abstract— Using high-intensity actinic light, the chlorophyll a fluorescence transient from HCO^-₃-depleted chloroplasts shows a rapid initial rise (O → I) followed by a slow phase (I → P). In the presence of HCO^-₃, the O → I rise is delayed but the I → P phase is much more rapid. Using low-intensity actinic light, the chlorophyll a fluorescence transient from 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU)-treated chloroplasts is delayed in the presence of HCO^-₃. Bicarbonate increases the amount of delayed light emission from chloroplasts given 10 s illumination with weak blue light (0·4 W/m²). DCMU greatly increases the amount of delayed light seen in the presence of HCO^-₃ under these conditions but decreases the amount seen in the absence of HCO^-₃. It is suggested that HCO^-₃ may somehow form or stabilize, in the dark, a number of reaction centers corresponding to the S₁ state in the model of B. Forbush, B. Kok and M. McGloin ( Photochem. Photobiol. 14, 307–321, 1971).     

GAMMA AND PULSE RADIOLYSIS STUDIES OF THE REACTION BETWEEN SUPEROXIDE IONS AND OXYHEMOGLOBIN-METHEMOGLOBIN SYSTEM

Abstract— The radiolytic studies of oxyhemoglobin or methemoglobin in neutral aerated aqueous solutions with formate ions, lead to three conclusions:
The oxidation of oxyhemoglobin by O^-₂ is not important. The observed low oxidation yield is probably due to the slow reaction with hydrogen peroxide produced by O^-₂ disproportionation.
The reduction of methemoglobin in γ radiolysis reaches a plateau which could be explained by structural considerations.
The reduction of methemoglobin by O^-₂ ions, if it occurs, is relatively slow: k = 1.4 × 10³ M ^-1 s^-1. But a problem remains concerning the spectral characteristics of the product.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

FUNCTIONAL DETERGENT PROBES OF MICELLE STRUCTURE. ABSORPTION, FLUORESCENCE AND QUENCHING MEASUREMENTS

Abstract— The absorption and fluorescence spectra of two functional ionic detergents containing an indole chromophore have been studied as micelle probes and have been compared to the corresponding spectra of 1-methylindole. The quenching of the fluorescence of the probes by NO^-₂ and Co²⁺ has also been investigated. Based on the results obtained, it is concluded that 1-methylindole is associated with the surface of HDTBr micelles, whereas it is located in the interior of SDS micelles. The chromophore of the indole detergents is considered to be located in the interior of HDTBr and SDS micelles.     

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.     

"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.     

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.     

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.     

ON THE REACTION OF SINGLET MOLECULAR OXYGEN WITH N-ALLYLTHIOUREA IN AQUEOUS SOLUTION

Abstract— Experiments on the photooxidation of N -allylthiourea, thiourea, and N-allylurea sensitized by the dye phenosafranine show that in N -allylthiourea the thiourea group is the site of singlet oxygen attack, while the allyl moiety neither reacts with nor quenches this metastable form of O₂ (in neutral aqueous solutions). Low concentrations of N^-₃ (a known quencher of singlet oxygen) strongly reduce the photooxidation of allylthiourea by a mechanism which apparently obeys simple competition kinetics. From these results the rate constant of the reaction between allylthiourea and singlet oxygen is obtained ( k = 4 × 10⁶ M ^-1 s^-1; pH = 7.1).     

WAVELENGTH-DEPENDENT QUANTUM YIELD OF OH RADICAL FORMATION FROM PHOTOLYSIS OF NITRITE ION IN WATER

Abstract. Photolysis (298- or SCN^- scavenging of OH and ³BP (benzophenone) actinometry. The significance of these results, and their dependence on pH, temperature, and [O₂], for the photolysis of nitrite in seawater is discussed briefly.     

THE ELECTROPHOTOLUMINESCENCE OF CHLOROPLASTS

Abstract— Delayed light emission emanating from preilluminated chloroplasts can be perturbed with pulsed DC electric fields (200–4000 V cm^-1), The perturbation produces a strong stimulation of chlorophyll luminescence. During the field perturbation the stimulated emission rises to a maximum, typically within 100μs. and then decays. Two kinetic components, R (rapid) and S (slow)†, are distinguished on the basis of their rise and decay times and their field-dependence. The R component increases exponentially at high fields, decays within 100–300μs during the field pulse and collapses with t _1/2= 15 μs at the end of the field pulse. The S component occurs at low fields, exhibits near saturation at 500 V cm^-1, decays with t _1/2 about 3 ms during the field pulse, and collapses with t _1/2= 38μs at the end of the field pulse. Studies using inhibitors, ionophores, electron donors and electron acceptors associate the R component with ion transport processes. The relation to electron transport associated with Photosystem II is discussed.     

CHEMICAL REACTIONS OF SUPEROXIDE ANION RADICAL IN APROTIC SOLVENTS

Abstract— While superoxide anion radical does not normally react with olefins, it does react with activated double bonds. Thus O^-₂ oxidatively cleaves certain α,β-unsaturated ketones such as chalcones and tetracyclone and electron poor olefins such as 1,1-dicyano and 1,1-dinitro olefins. Similarly O^-₂ can react with substituted nitrobenzenes to yield the corresponding nitrophenols. EPR and oxygen labeling (KO³⁶₂) experiments confirm an electron transfer mechanism.     

MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE a STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON spIN RESONANCE SPECTROSCOPY

Abstract Phcophorbide a (PPa), a causal substance of food intoxication, when excited by exposure to light wavelengths of over 600 nm, caused the photohemolysis of goat erythrocytes in proportion to the incubation time of the cells. The addition of N-₃, an effective scavenger of ¹O₂, to the medium markedly inhibited the hemolysis of erythrocytes in a concentration-dependent manner, whereas the addition of superoxide dismutase (SOD) and catalase, inhibitors of O^-₂ and H₂O₂ generation, respectively, to the medium had little effect on it.
Methods for converting ¹O₂ to a nitroxide radical by 2,2,6,6-tetramethyl-4-piperidone (TMPD) and for trapping O^-₂ and OH by 5,5-dimethyl-l-pyrroline-A'-oxide (DMPO) were employed to observe directly these activated oxygens by electron spin resonance (ESR). The methods provided evidence that only ¹O₂, was produced by PPa, which was excited by light wavelengths of over 600 nm. Both the addition of N₃ to the solution and the removal of oxygen from the solution inhibited the generation of ¹O₂.
These results led us to conclude that ¹O₂ was mainly responsible for the hemolysis of erythrocytes by photoexcited PPa.     

BIMANES—26. AN ELECTRON TRANSFER REACTION BETWEEN PHOTOSYSTEM I1 AND MONOBROMOBIMANE INDUCES STATIC CHLOROPHYLL a QUENCHING IN SPINACH CHLOROPLASTS

Abstract— Monobromobimane in chloroplasts lowers both the quantum yield of system II photochemistry and the yield of chlorophyll a fluorescence. Illumination of the chloroplasts in the presence of monohromobimane is an absolute prerequisite to the manifestation of this phenomenon, which proceeds via the Photosystem II intermediate, the semiquinone radical anion, Q_A-. The latter transfers an electron to monobromobimane to yield an anion radical (mBBr^·), which may either lose bromide ion to yield a reactive radical (mB^·), or acquire a proton and undergo further reduction, eventually forming syn-(methyl, methyl) bimane. In turn, mB reacts with the protein of the light-harvesting complex, to form a product which acts as static excitation energy quencher in the chlorophyll pigment bed of photosystem 11. Polarographic reduction of monobromobimane shows an adsorption wave at O V and two reduction waves. Prolonged reduction in water at -0.5 V yields syn-(methyl, methyl) bimane (which is further reduced at more negative potentials) and bromide ion. Thus, both electrochemical and chloroplast-induced reduction produce syn-(methyl, methyl) bimane. Monobromobimane may then serve as a Photosystem II activated probe in elucidating the conformation of intrinsic thylakoid membrane polypeptides.     

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.     

DYE-SENSITIZED PHOTOREDUCTION OF METHYL VIOLOGEN

Abstract— The kinetics of the proflavine-sensitized photoreduction of methyl viologen (MV²⁺) to the blue radical cation (MV^.+) are presented. The triplet excited state of proflavine accepts an electron from EDTA to form the singly-reduced species of proflavine (PH^.); this species donates an electron to either the oxidized (MV^.+) or the singly–reduced (MV^.+) species of the bipyridyl. MV²⁺ can be reoxidized by oxygen to MV²⁺ but is decomposed by H₂O₂. The doubly-reduced form can not be reoxidized either by oxygen or by peroxide. Potassium iodide inhibits the photoreduction of MV²⁺ by competing with it as reactant for the singly-reduced form of proflavine (PH^.). The mechanism presented may be analogous to that occurring in the reduction of MV²⁺ by illuminated spinach chloroplasts; its herbicidal action can not be ascribed to the formation of peroxides.     

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.     

Ultraviolet Radiation Effects on a Microscopic Green Alga and the Protective Effects of Natural Dissolved Organic Matter

Abstract— The population and photosynthetic responses of a microscopic green alga ( Selenastrum capricornutum ) to realistic levels of UV radiation (UVA and UVB) were assessed in natural lake waters of different dissolved organic carbon (DOC) concentration. Specific growth rates and photosynthetic competence (as reflected by F_v/F_m [measure of maximal quantum efficiency of photosystem II] and t_1/2 [estimate of electrons transported to the plastoquinone pool] measured by in vivo variable chlorophyll a fluorescence) were compared between two exposure levels of UVR and two concentrations of DOC (2.5 mg C L⁻¹ 7.7 mg C L⁻¹). Exposure periods of 6–9 days (five to nine generations) were used. Exposure to UVA primarily affected the efficiency of photosystem II, as evidenced by significant decreases of F_v/F_m but not growth rates or t_1/2 Exposure to UVB, in the presence of UVA, did not cause significant additional decreases of F_v/F_m but did diminish growth rates. In the low DOC water, t_1/2 was also diminished, suggesting different proximate sites of action from those for UVA. The high DOC water decreased the effective exposure to both UVA and UVB and diminished the negative impact of UV radiation on the cells, but the apparent protection was not explicable solely by the shading action of the DOC. Control values for F_v/F_m, growth rates and t_1/2 were all lower in the high DOC water, suggesting a negative side effect to the apparent protective action of the DOC against UVB.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.