Methyl Viologen-Dependent Cyclic Electron Transport in Spinach Chloroplasts in the Absence of Oxygen

The methyl viologen (MV)-dependent, linear electron flow fromPS II to PS I was severely blocked in intact or broken, uncoupledchloroplasts when oxygen was removed from the suspension medium,as revealed by measurements of chlorophyll fluorescence andthe rate of photoreduction of MV. Kinetics of the reductionof pre-oxidized P700 by a saturating light pulse showed thatreduced MV in the absence of oxygen re-reduces P700⁺ via theintersystem electron transport chain. Since the re-reductionof P700⁺ was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone,the MV-mediated cyclic electron flow, in contrast to the phenazinemethosulphate-catalyzed one, involves the plastoquinone pool.However, 2-n-heptyl-4-hydro-xyquinoline-N-oxide, 2-n-nonyl-4-hydroxyquinoline-N-oxideand antimycin A did not inhibit the MV-mediated flow. Thus,the inhibition of the linear electron flow in chloroplasts underanaerobic conditions suggested the overreduction of the plastoquinonepool as a result of the MV-mediated cyclic flow (Received February 13, 1990; Accepted March 31, 1990)     

Enhanced ferredoxin-dependent cyclic electron flow around photosystem I and α-tocopherol quinone accumulation in water-stressed <Emphasis Type="Italic">ndhB</Emphasis>-inactivated tobacco mutants

Dissipation mechanisms of excess photon energy under water stress were studied in ndhB-inactivated tobacco (Nicotiana tabacum cv. Xanthi) mutants, which are impaired in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI. Relative leaf water content and net CO₂ assimilation decreased to 30% and almost zero, respectively, after 11-day water stress in the mutant and wild type plants. Similar reductions in PSII activity (by ca. 75%), and increases in malondialdehyde (by ca. 45%), an indicator of lipid peroxidation, were observed in both the plant groups when subjected to water stress. The stressed mutant and wild type plants showed similar P700 redox kinetics, but only the stressed mutant demonstrated an enhanced operation of the antimycin A-sensitive, ferredoxin-dependent cyclic electron flow around PSI, as indicated by a transient increase in chlorophyll fluorescence after turning off of actinic light. Further, the stressed mutant showed higher oxidation of -tocopherol to -tocopherol quinone, as compared with that in the stressed wild type. Thus, a deficiency in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI does not lead to oxidative damage because the mutant compensates for this deficiency by activating alternative dissipating routes of excess photon energy, such as up-regulation of ferredoxin-dependent cyclic electron flow around PSI and increased accumulation of -tocopherol quinone.     

Generation of Superoxide Anion and Localization of CuZn-Superoxide Dismutase in the Vascular Tissue of Spinach Hypocotyls: Their Association with Lignification

The sites of generations of superoxide anions and hydrogen peroxidein cross sections of hypocotyls from spinach seedlings werelocated by staining with nitroblue tetrazolium (NBT) and withstarch-iodide, respectively. Formazan, produced upon the reductionof NBT by superoxide, was observed mainly in the vascular tissueonly in the presence of inhibitors of CuZn-superoxide dismutase(CuZn-SOD), and its formation was suppressed under anaerobicconditions. Thus, NBT was reduced to formazan specifically bythe superoxide anions generated in vascular tissue. The reductionof NBT was suppressed by inhibitors of NAD(P)H oxidase, butneither by cyanide nor azide, indicating the involvement ofNAD(P)H oxidase in the generation of superoxide anions in thevascular tissue. Starch-I₂ complex also was formed in the vasculartissue, but not in the presence of either the CuZn-SOD inhibitoror the NAD(P)H oxidase inhibitor, indicating that the hydrogenperoxide is produced via the catalytic disproportionation withCuZn-SOD of the superoxide generated by NAD(P)H oxidase. Generationsof superoxide anions and hydrogen peroxide in the vascular tissuewere particularly apparent in the xylem and associated withthe sites of distribution of CuZn-SOD as determined by an immunohistochemicalmethod, and also with the location of lignin as determined bythe phloroglucin-HCI reaction. ⁴Present address: Chemical Research Laboratories, Toray IndustriesInc., 9–1 Oe-cho, Minato-ku, Ngagoya, 455 Japan     

Spinach chloroplasts scavenge hydrogen peroxide on illumination

Intact spinach chloroplasts isolated by the modified silicasol density centrifugation scavenged H₂O₂ on illumination ata rate about 3-fold that of bicarbonate-dependent O₂-evolution.Accompanying the disappearance of 1 mole of H₂O₂ is the evolutionof a half mole of O₂. The photoscavenging of H₂O₂ was inhibitedby 3(3,4-dichloro-phenyl)-l, l-dimethylurea, cyanide and azide.These results indicate that in chloroplasts H₂O₂ is reducedto H₂O by a cyanide and azide-sensitive peroxidase using a photoreductantas an electron donor. (Received July 4, 1980; )     

Isolation of the D1 Protein and a 28-kDa Fragment of the D2 Protein from Spinach Photosystem II Membranes

The 32-kDa D1 protein, which contained no lysine in spinachchloroplasts, as deduced from its DNA code, was isolated byhigh-performance gel permeation chromatography in the presenceof 0.1% SDS and 4 M urea. Three proteins of the photosystemII reaction center complex have a molecular mass of 30–35kDa, and two, the D2 protein and the peripheral 33-kDa protein,were severed into peptide fragments by Achromobacter lysyl endopeptidase(EC 3.4.14.50 [EC] ) before the chromatography. The isolated D1 proteindid not contain chlorophylls and pheophytins but had an absorptionmaximum at 265 nm probably due to bound plastoquinone. A peptidefragment of 28 kDa from the D2 protein was also isolated fromspinach photosystem II membranes and the wheat photosystem IIreaction center. Antibodies raised against the 28-kDa peptidefrom wheat bound to the 34-kDa D2 protein, which suggested thatthis peptide was the largest sequence of Aspl4-Lys265. The fragmentof wheat D2 protein showed absorption maxima at 413 and 682nm attributable to bound pheophytin that probably had been convertedfrom chlorophyll a during the isolation process. (Received June 29, 1987; Accepted October 21, 1987)     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings V. Protein Synthesis in Vivo

Incorporation of ¹⁴C-amino acids into proteins in radish cotyledonswas suppressed by 4-thiouridine (4SU) culture. The inhibitoryeffect of 4SU was similar to that of chloramphenicol. 4SU culturedid not reduce the content of ferredoxin (Fd) and the labelinginto Fd significantly, but it did decrease the content and thesynthesis of ribulose bisphosphate carboxylase (RuBPCase). Thesynthesis of thylakoid chlorophyll-proteins I and II also wasinhibited by 4SU culture. In 4SU-cultured seedlings, the ratioof labeling into the large and small subunits of RuBPCase andthat into the two chlorophyll-proteins were the same as thosein the controls grown without 4SU. (Received September 29, 1980; Accepted January 27, 1981)     

Oxidation Rate of Monomeric Radish Cytochrome f with Plastocyanin

The reaction rate of reduced monomeric cytochrome f with oxidizedplastocyanin, both purified from Japanese radish, was determinedby a stopped-flow method. The oxidation rate constant was 6.0x 10⁷ M¹sec¹ at pH 7.0 and 25°C, which is slightly higherthan the value reported by Wood [(1974) Biochim. Biophys. Acta357 : 370] for oligomeric parsley cytochrome f Thermodynamicparameters also were determined to be 56 KJ M^–1 for activationenthalpy and 90 J M^–1 K^–1 for activation entropy.Neither a pH from 6 to 9 nor the addition of NaCl, polylysine,histone or polyaspartate affected the rate constant. ¹Present address: The National Institute for Environmental Studies,Yatabe, Ibaraki 305, Japan. (Received October 9, 1980; Accepted November 17, 1980)     

Purification of cytochrome f from Japanese-radish leaves

Cytochrome f was purified (A_420.5/A₂₇₃, 2.0) from acetone extractsof Japaneseradish leaves without use of detergent. By gel filtrationwidi Sephadex G-100 the molecular weight was estimated to be33,000 daltons. (Received September 17, 1974; )     

Paraquat Resistant Tobacco Calluses with Enhanced Superoxide Dismutase Activity

Seven paraquat resistant calluses of tobacco (Nicotiana tabacumL. cv. Samsun) were obtained by three successive screeningsof protoplast-derived calluses on a paraquat containing medium.Superoxide dismutase (SOD) activity of the resistant calluseswas 14- to 159-fold that of the leaf cells on protein basis.Paraquat-resistant calluses, however, showed little increasein catalase and peroxidase activities. More than 90% of SODactivity in the resistant calluses was inhibited by KCN, aswas the SOD activity in leaves, indicating that the major SODin the callus appears to be the Cu, Zn containing enzyme. Thecallus cells, however, expressed the immunologically distinguishedSOD isozyme from the enzyme in the leaves. (Received April 23, 1984; Accepted August 6, 1984)     

Ascorbate Peroxidase in Tea Leaves: Occurrence of Two Isozymes and the Differences in Their Enzymatic and Molecular Properties

Two isozymes of ascorbate (AsA) peroxidase were found in tealeaves, and one of them (AsA peroxidase II) was purified tohomogeneity, as judged by polyacrylamide gel electrophoresis.AsA peroxidase II is a monomer with a molecular weight of 34,000and contains protoheme, but it is not a glycoprotein. The enzymeshowed a Soret peak at 409 run and at 420 nm when oxidized andreduced, respectively, with an a-band at 556 nm. The oxidizedenzyme showed two small peaks at 478 nm and 530 nm. The peakat 478 nm disappeared when the enzyme was inactivated by depletionof AsA or by the addition of cyanide. Antibody raised againstAsA peroxidase II from tea did not cross-react with guaiacolperoxidase from spinach, and antibody against the guaiacol peroxidasedid not with AsA peroxidases from tea leaf. The amino acid compositionand amino acid sequence of the amino-terminal region of AsAperoxidase II were determined. Little homology in terms of aminoacid sequence was found between AsA peroxidase II and variousguaiacol peroxidases. The enzymatic and molecular propertiesof the two isozymes showed distinct differences with respectto molecular weight, sensitivity to AsA-depletion, specificityfor the electron donor, and other enzymatic properties. (Received April 13, 1989; Accepted July 25, 1989)