Dark Induction of the Non-Photochemical Quenching of Chlorophyll Fluorescence by Acetate in Chlamydomonas reinhardtii

Addition of acetate to a suspension of Chlamydomonas reinhardtiicells in darkness induced transient and biphasic non-photochemicalquenching of Chl fluorescence (qN) due to ApH-dependent down-regulationof PSII and the transition from state 1 to state 2. We proposethat acetate-induced stimulation of the chlororespiratory electronflow triggers the regulation of PSII. (Received December 26, 1995; Accepted March 25, 1996)     

Anticonvulsant Effectiveness and Neurotoxicity Profile of 4-butyl-5-[(4-chloro-2-methylphenoxy)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (TPL-16) in Mice

Neurochemical Research - Protective (antiseizure) effects of 4-butyl-5-[(4-chloro-2-methylphenoxy)-methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (TPL-16) and acute neurotoxic effects were...     

Photoproduction of the Azidyl Radical from the Azide Anion on the Oxidizing Side of Photosystem II and Suppression of Photooxidation of Tyrosine Z by the Azidyl Radical

Azide ions inhibited O₂ evolution in PSII membranes from spinachin a time-dependent manner in the light until all activity disappeared.Illumination in the presence of azide (azide-phototreatment)irreversibly inhibited the following processes: (1) both theoxidation of water and the electron transport between the redox-activetyrosine 161 of the D1 protein (Y_Z) and the secondary quinoneelectron acceptor (Q_B) site, to the same extent; (2) the donationof electrons to the primary quinone electron acceptor (Q_A),as measured by monitoring the maximum variable fluorescenceof Chl; and (3) the photoproduction of the Y_Z radical (Y). Thus,the primary site of inhibition appeared to lie between Y_Z andQ_A. On illumination of Tris-treated PSII membranes in the presenceof azide, production of the azidyl radical was observed by spin-trappingESR. Yield of Y in Tris-treated membranes on illumination wassuppressed by azide. Electron transport from Y_Z to Q_B in Tris-treatedmembranes was inhibited only when the azidyl radical was photoproduced,and it was inhibited more rapidly than it was in the oxygenicPSII membranes. These results indicate that the azidyl radicalwas produced via a univalent oxidation of azide by Y and thatit irreversibly inhibited the electron transport from Y_Z toQ_A in Tris-treated membranes. Although the azidyl radical wasundetectable in the oxygenic PSII membranes, probably due tosteric interference by the peripheral proteins of water-oxidizingcomplex with the access of the spin-trapping reagent to theproduction site of the radical, the participation of the azidylradical in the inhibition of the oxygenic PSII membranes issuggested since simultaneous occurrence of both electron transportand azide was required for the inhibition. Possible inhibitorymechanisms and the target sites of azidyl radical are discussed. (Received April 21, 1995; Accepted July 3, 1995)     

Quenching Analysis of Chlorophyll Fluorescence by the Saturation Pulse Method: Particular Aspects Relating to the Study of Eukaryotic Algae and Cyanobacteria

The principles of chlorophyll fluorescence quenching analysisby the saturation pulse method are outlined with emphasis onparticular aspects encountered in the study of eukaryotic algaeand cyanobacteria. Major differences of these photosyntheticorganisms with respect to higher plant leaves, for which quenchinganalysis originally was developed, are very rapid inductionof O₂-dependent electron flow, close interaction between photosyntheticand respiratory metabolism, dark reduction of the plastoquinonepool and pronounced state transitions of energy distributionbetween the two photosystems. It is shown that the use of 25–50ms pulses of saturating light for determination of maximal fluorescenceyield is advantageous, in contrast to the 0.5–2 s pulsescommonly used with higher plants. The shorter pulses are lessinvasive with respect to the induction of energising electronflow which can induce non-photochemical quenching and statechanges. In particular, short saturation pulses are essentialto study true dark changes of fluorescence yield. As an example,the induction of pronounced quenching of maximal fluorescencein Chlamydomonas by dark-anaerobic incubation is demonstrated.Analysis of the rapid rise kinetics upon onset of saturatinglight reveals two major phases, O-I₁ and I₁-I₂, with distinctlydifferent properties. Arguments are put forward that an assessmentof maximal fluorescence yield with single turnover saturatingflashes is problematic, as there is a type of photochemicalquenching, the elimination of which during the I₁-I₂ phase requiresmultiple turnovers at photosystem II. Furthermore, the variablefluorescence represented by the two phases is affected differentlyby non-photochemical quenching. It is shown that dark-anaerobicquenching in Chlamydomonas as well as state 2 quenching in Synechocystisare correlated with a preferential suppression of the I₁-I₂phase. Experiments with Synechocystis are presented which demonstratethe potential of saturation pulse quenching analysis for thestudy of reversible state changes. The mutant M55 of Synechocystis6803 appears to be "locked" in state 1. ¹Present address: Lehrstuhl Botanik I, Mittlerer Dallenbergweg64, D-97082 Würzburg, Germany     

CuZn-Superoxide Dismutases in Rice: Occurrence of an Active, Monomeric Enzyme and Two Types of Isozyme in Leaf and Non-Photosynthetic Tissues

Rice leaves and seed embryos contain four isozymes of CuZn-superoxidedismutase (SOD) and two isozymes of Mn-SOD. CuZn-SOD I is amajor enzyme in leaves, but not in embryos or etiolated seedlings.CuZn-SODs II,III and IV were found in the embryos but were alsofound as minor isozymes in leaves. CuZn-SODs I, II and IV were purified to homogeneity from riceleaves. CuZn-SODs I and II had similar properties with respectto molecular weight, dimeric structure, absorption spectrumand metal content, but their amino acid compositions differedfrom each other. The absorption spectrum of CuZn-SOD IV wassimilar to that of isozymes I and II, but this enzyme was amonomer with a molecular mass of 1.7 kDa. Antibody against CuZn-SODI from rice did not cross-react with isozymes II and IV. Antibodiesagainst CuZn-SOD from spinach leaves cross-reacted with isozymeI but not with isozymes II, III and IV. By contrast, the antibodiesagaist CuZn-SOD from spinach seeds cross-reacted with isozymesII, III and IV but not with isozyme I. Thus, the isozyme thatis expressed mainly in leaves (CuZn-SOD I) and the isozymesexpressed mainly in non-photosynthetic tissues (CuZn-SODs II,III, IV) are immunologically distinct. (Received October 7, 1988; Accepted January 27, 1989)     

CuZn-Superoxide Dismutases from the Fern Equisetum arvense and the Green Alga Spirogyra sp.: Occurrence of Chloroplast and Cytosol Types of Enzyme

CuZn-superoxide dismutase (SOD) from horsetail (Equisetum arvense)was purified to a crystalline state and that from pond scum(Spirogyra sp.) was purified to a mixture of three isozymes.The purified CuZn-SODs from the fern and the green alga showsimilar properties to those of the angiosperm and mammalianenzymes with respect to molecular weight, subunit structure,absorption spectrum, circular dichroism spectrum and the effectof modification of the arginine residues by 2,3-butanedioneon activity. Horsetail and pond scum contained three isozymeseach of CuZn-SOD. These isozymes are divided to two types: onetype gave a cross-reaction with antibody raised against chloroplast-typeCuZn-SOD from spinach and other type cross-reacted with antibodyraised against cytosol-type CuZn-SOD from spinach. Thus, itappears that the divergence of the chloroplast and cytosol typesof CuZn-SOD started at a very early stage in the molecular evolutionof this enzyme. (Received January 30, 1989; Accepted April 19, 1989)     

Electron Donation from Cyclic and Respiratory Flows to the Photosynthetic Intersystem Chain is Mediated by Pyridine Nucleotide Dehydrogenase in the Cyanobacterium Synechocystis PCC 6803

The redox kinetics of P700 induced by far-red light and a pulseof strong white light in wild type cells were compared withthose in NAD(P)H dehydrogenase (NDH)-defective mutants of thecyanobacterium Synechocystis PCC 6803. The wild type cells showedthe electron donation from the respiratory donor and the photoreductantgenerated in PS I to P700⁺ through the plastoquinone, whichis mediated by a Hg²⁺-sensitive enzyme. The NDH-defective mutantcells, however, did not show the electron transfer to P700⁺through the plastoquinone from both the photoreductant in PSI and cytosolic electron donors using pyndine nucleotides asan intermediate. Thus, NDH appears to be the site of main entryof electrons into the plastoquinone pool in the NAD(P)H-mediatedcyclic electron flow and the respiratory electron flow in Synechocystis. (Received August 31, 1992; Accepted October 1, 1992)     

Superoxide dismutases from kidney bean leaves

Three isozymes of superoxide dismutase were found in the solubleextract of kidney bean leaves. Two of them, purified to nearhomogeneous states, were inhibited by cyanide and by the antibodyto spinach Cu, Zn-superoxide dismutase. Thus, both isozymeswere the Cu and Zn containing enzyme that has a molecular weightof 32,000 and is composed of two subunits of equal size. Anotherisozyme was insensitive to cyanide and to the antibody, butwas inactivated by acetone or heating. The cyanideinsensitiveisozyme was not inactivated by H₂O₂ showing that this isozymeis Mn-superoxide dismutase. (Received June 13, 1979; )     

Photoautotrophy and the photosynthetic potential of chlorophyllous cells in mixotrophic cultures

The relationship between the photoautotrophy of cultured plantcells and their photosynthetic potentials in mixotrophic cultureswas investigated. All chlorophyllous cells took up oxygen inthe dark, but illumination immediately reduced the uptake. Highlychlorophyllous scotch broom and tobacco cells actively evolvedphotosynthetic oxygen when NaHCo₃ was added, but chlorophyllousamur cork-tree cells showed very little evolution. The measurementof photosynthetic oxygen evolution in chlorophyllous cells revealeda parallel between the photosynthetic potential and photoautotrophy.Activities of photosystems I and II and of ribulose-1,5-bisphosphatecarboxylase showed that the low photosynthetic activity of amurcork-tree cells was mainly due to the low activity of photosystemII. (Received September 1, 1978; )     

NAD(P)H Dehydrogenase-Dependent Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechocystis PCC 6803: a Study of Dark-Starved Cells and Spheroplasts

Electron donation to P700⁺ through plastoquinone in the intersystemchain from both respiratory substrates and the photoreductantsin PSI has been shown to be mediated by the NAD(P)H-dehydrogenasecomplex (NDH) in Synechocystis PCC 6803 cells [Mi et al. (1992)Plant Cell Physiol. 33: 1233]. To confirm the participationof NDH in the cyclic electron flow around PSI, the redox kineticsof P700 and Chi fluorescence were analyzed in cells rendereddeficient in respiratory substrates by dark starvation and inspheroplasts. Dark-starved cells showed a high steady-state level of P700⁺under far-red (FR) illumination and the plastoquinone pool wasin a highly oxidized state. An NDH-defective mutant consistentlyshowed a high level of P700 oxidation under FR before and afterthe dark starvation. Donation of electrons either from exogenousNADPH or from photoreduced NADPH⁺ to the intersystem chain viaplastoquinone was demonstrated using spheroplasts from wild-typecells, but not those from the NDH-defective mutant, as monitoredby following changes in the kinetics of Chi fluorescence andthe redox state of P700. The electron flow to PSI via plastoquinone,mediated by NADPH, was sensitive to rotenone, Hg²⁺ ions and2-thenoyltrifluoroacetone, inhibitors of mitochondrial NDH andsuccinate dehydrogenase, but not to antimycin A. The pool sizeof electrons that can be donated to P700⁺ from the cytosol throughthe intersystem chain increased with increasing duration ofillumination time by actinic light and was sensitive to rotenonein both wild-type cells and spheroplasts, but no such resultswere obtained in the NDH-defective mutant of Synechocystis 6803.The results support our previous conclusion that NDH is a mediatorof both respiratory electron flow and cyclic electron flow aroundPSI to the intersystem chain in the cyanobacterium Synechocystis. (Received August 20, 1993; Accepted November 22, 1993)