Effects of 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) on Light Induced and Glycolytic Phosphate Uptake in Scenedesmus

The effects of DBMIB on photophosphorylation and glycolysis in Scenedesmus obtusiusculus Chod. were investigated by measuring the uptake of inorganic phosphate. To analyze the effects of DBMIB on the different energy coupling possibilities in open chain and cyclic photophosphorylation, DBMIB was given to the algae in narrow concentration intervals between 10^?6M to 10^?4M, either alone, or in combination with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) or desaspidin. DBMIB inhibits non-cyclic as well as cyclic photophosphorylation in Scenedesmus. However, the DCMU resistant photophosphorylation reactions are less sensitive to DBMIB than the open chain photophosphorylating system in non-DCMU treated cells. Low concentrations of DBMIB even released a part of the DCMU inhibition. Experiments with combinations of DBMIB and desaspidin also indicated that cyclic photophosphorylation is less sensitive to DBMIB than non-cyclic. The inhibition of DCMU resistant cyclic phosphorylation by DBMIB, which is a competitive inhibitor of quinones, indicated a participation of plastoquinones in this type of energy coupling as well as in the non-cyclic and DCMU-sensitive processes. The cyclic and the non-cyclic photophosphorylation pathways probably use different parts of the plastoquinone pool. For the purpose of the experiments, it was necessary to produce data for the effect of DBMIB (10^?6–10^?4M) on glycolysis. The highest concentration gave 50% inhibition.     

Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat

Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448–455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the latter was largely ineffective in reducing the cytosolic ATP/ADP ratio. Thus, the results show that the antimycin-sensitive electron transport is, potentially, equally active in light and darkness. In addition, they indicate that antimycin-insensitive electron transport in mitochondria (alternative pathway) does not significantly contribute to the cytosolic energy state.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropy-p-benzoquinone) - DCMU dichlorophenyldimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy-phenylhydrazone - SHAM sancylhydroxamic acid     

Clotrimazole对菠菜叶绿体光合磷酸化和电子传递的抑制作用

10μmol/的clotrimazole不仅抑制光合磷酸化活力,而且抑制各种类型的电子传递,是一个典型的电子传递抑制剂。经过它对叶绿体放氧,荧光和毫秒延迟发光影响的比较研究表明:clotri—mazole在光合电子传递链上的作用部位在Q与PQ之间,即与DGMU的作用部位相同或相近。     

The effect of p-phenylenediamine and dibromothymoquinone on the postosynthetic electron transport and proton pump activities of isolated barley chloroplats

The effect of p-phenylenediamine and dibromothymoquinone (DBMIB) on photosynthetic electron transport was studied using thylakoids from barley chloroplasts. p-Phenylenediamine (0.1 mm) converted the light saturation curve for electron transport between water and ferricyanide from a hyperbolic one saturating at low light intensity into a linear one which was not saturated at any light intensity studied. DBMIB at a concentration of 1 μm had little inhibitory effect on the basal electron transport activity of coupled thylakoids in either the presence or the absence of 0.1 mmp-phenylenediamine. However, activity was inhibited by DBMIB after uncoupling with 60 mm methylamine. These results may indicate that there is a rate-limiting step in electron transport at or after plastoquinone which is circumvented by phenylenediamine. Its appearance or elaboration could explain the conversion of a linear light saturation curve to a hyperbolic one during chloroplast development. The movement of protons into thylakoids upon illumination was reduced about 50% by 1 μm DBMIB, a concentration which maximally inhibits ferricyanide-Hill activity after uncoupling. Fifty percent inhibition occurred irrespective of whether or not phenylenediamine was included in the reaction mixture. Most of the residual DBMIB-insensitive pH change was inhibited by 3 μm 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), suggesting that photosystem II was required for the latter activity. In the presence of both DBMIB and DCMU, proton pump activity could be reestablished by including isoascorbate and p-phenylenediamine in the reaction mixture.     

Photomovement of the red alga Porphyridium cruentum (Ag.) naegeli I. Photokinesis

Photokinesis of the red alga Porphyridium cruentum was studied with the aid of a population method. Because of the slow spreading velocity (0.35 m/min) the duration of the experiments was 7 days in general. According to the white light illuminance-response curve the zero threshold of photokinesis lies below 10 lx and the optimum around 10,000 lx. With further increasing illuminance the photokinetic effect decreases, reaching zero at about 100,000 lx. The action spectrum indicates that the photokinetically active radiation is absorbed by photosynthetic pigments, namely the biliproteins B-phycoerythrin, R-phycocyanin and allo-phycocyanin, as well as by chlorophyl a, although the photokinetic effect of blue light is relatively low. From the action spectrum and the results of inhibitor experiments with DCMU, DBMIB and DSPD it is concluded that the photokinetic effect is due to an additional ATP supply from non-cyclic and/or pseudo-cyclic photophosphorylation to the motor apparatus.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) - DSPD Disalicylidenepropanediamine-(1,3)     

Investigations on the phototactic orientation of Anabaena variabilis

Phototaxis of the blue-green alga Anabaena variabilis was studied using both population method and observation of single trichomes by microscope. The trichomes react positively at low and negatively at high illuminance. The inversion point lies at about 1000 1x. The action spectrum of positive phototaxis indicates that the photosynthetic pigments chlorophyll a, C-phycocyanin and allo-phycocyanin are involved in the absorption of the active light. The same range of wavelengths is active in negative phototaxis, but in addition, wavelengths between 500 and 560 nm and between 700 and 750 nm are also effective. Obviously pigments of unknown chemical nature are sharing in light absorption. Two alternatives are discussed. Since inhibitors of photosynthesis such as DCMU and DBMIB do not affect phototactic orientation, a direct coupling of phototaxis with photosynthesis can be excluded.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) Presented in part at the International Symposium on Photosynthetic Prokaryotes: August 22–28, 1976, Dundee, Scotland     

Alternative Electron Transports Participate in the Maintenance of Violaxanthin De-Epoxidase Activity of Ulva sp. under Low Irradiance

The xanthophyll cycle (Xc), which involves violaxanthin de-epoxidase (VDE) and the zeaxanthin epoxidase (ZEP), is one of the most rapid and efficient responses of plant and algae to high irradiance. High light intensity can activate VDE to convert violaxanthin (Vx) to zeaxanthin (Zx) via antheraxanthin (Ax). However, it remains unclear whether VDE remains active under low light or dark conditions when there is no significant accumulation of Ax and Zx, and if so, how the ΔpH required for activation of VDE is built. In this study, we used salicylaldoxime (SA) to inhibit ZEP activity in the intertidal macro-algae Ulva sp. (Ulvales, Chlorophyta) and then characterized VDE under low light and dark conditions with various metabolic inhibitors. With inhibition of ZEP by SA, VDE remained active under low light and dark conditions, as indicated by large accumulations of Ax and Zx at the expense of Vx. When PSII-mediated linear electron transport systems were completely inhibited by SA and DCMU, alternative electron transport systems (i.e., cyclic electron transport and chlororespiration) could maintain VDE activity. Furthermore, accumulations of Ax and Zx decreased significantly when SA, DCMU, or DBMIB together with an inhibitor of chlororespiration (i.e., propyl gallate (PG)) were applied to Ulva sp. This result suggests that chlororespiration not only participates in the build-up of the necessary ΔpH, but that it also possibly influences VDE activity indirectly by diminishing the oxygen level in the chloroplast.     

质体醌库和细胞色素b6f参与调控蓝细菌 Synechocystis sp. PCC 6803的状态转换

用调制叶绿素荧光研究了对苯醌(1,4-benzoquinone,BQ)和二溴百里香醌(2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone,DBMIB)对蓝细菌Synechocystissp.PCC6803状态转换的作用。BQ和DBMIB是质体醌(PQ)的类似物,两者均可充当PQ的电子受体。其中,DBMIB能够和细胞色素b6f的Qo位点特异结合。在没有作用光的情况下,BQ诱导暗适应的蓝细菌进入状态Ⅰ;相反,DBMIB诱导Syne鄄chocystis6803向状态Ⅱ转换。据此提出,在生理状态下蓝细菌根据PQ库的氧化还原状态调节状态转换;细胞色素b6f参与此调控过程。     

Light-Induced Phosphate Binding in Relation to Photophosphorylation and Levels of ATP, ADP and AMP in the Green Alga Scenedesmus

Synchronous cultures of Scenedesmus obtusiusculus Chod. were starved for phosphorus for 48 h. Such cells develop an efficient mechanism for phosphate binding which is very sensitive to metabolic inhibitions. Phosphate binding, fluctuations in the ATP pool during dark-light-dark transitions, and steady state levels of ATP, ADP and AMP were studied. The experiments were carried out in a CO₂-free N₂ atmosphere. DCMU, phloridzin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) were used as inhibitors of photophosphorylation. Light-induced phosphate uptake was inhibited to various extents by all the inhibitions. The dark-light-dark transition experiments show that neither the light-induced increment in ATP nor the decrease at darkening are affected by DCMU, but DBMIB and phloridzin inhibit both processes. DCMU seems to affect the regulation of the ATP pool size. The steady state levels of the adenylate pools were almost the same in the light as in the dark, and they were also little sensitive to the inhibitors. In unpoisoned cells in the light the steady state ATP/ADP ratio was 1.7 and the energy charge was 0.66. The rates of phosphate binding are not correlated to any of the adenylate parameters studied. This is probably due to the diverse effects of the inhibitors on light-stimulated production of reducing equivalents, photophosphorylation and transfer of energy from the chloroplast to the cytoplasm.     

Spectrophotometric measurement of plastoquinone photoreduction in the blue-green alga,Phormidium uncinatum

The redox state of plastoquinone was measured in vivo in the blue-green alga, Phormidium uncinatum by means of a double beam UV-spectrophotometer. The difference in absorbance of the oxidized and the reduced forms of plastoquinone was amplified, and stored and averaged in a computer. The redox state was changed by two alternating actinic light beams. When one actinic wavelength was kept constant at 700 nm (PSI) variation of the other yielded an action spectrum representing photosystem II. The inhibitors of the photosynthetic electron transport chain, DCMU and DBMIB, reduced the difference in absorbance between the oxidized and reduced forms of plastoquinone.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea     

A new way to monitor by-pass restorations of electron transport in inhibited chloroplasts by cyclic electron flow cofactors--a study by modulated fluorimetry.

Inhibition of electron transport in broken chloroplasts by DBMIB, under light-limiting conditions, is shown to be bypassed by PMS in a manner similar to the known effects of the phenylenediamine derivatives TMPD and DAD. These bypasses were demonstrated and further studied by modulated fluorimetry, monitoring DBMIB inhibition by the shift of the steady-state fluorescence towards the Fm level and the release of inhibition by a reverse shift together with establishment of a quenching effect by background far-red light. Comparative studies were also made with electron transport blocked by DCMU or BNT. A weak bypass by TMPD and a weaker one by PMS of the block created by DCMU was observed by modulated fluorimetry. The block created by BNT is similarly shown to be bypassed by TMPD but hardly or not at all by PMS. Bypass effects persisted even in the presence of ascorbate. It appears that, following reduction of the different cofactors by ascorbate in the stroma side, illumination caused the accumulation of a pool of oxidized cofactor molecules in the lumen, which is able to mediate electron transport between reduced plastoquinone and plastocyanin or P-700. The existence and the size of this pool were found to depend largely on the internal pH at the lumen, presenting an artificial system in which electron flow is controlled by the lumenal pH. The bypassing electron transport in the presence of DBMIB presumably avoids the participation of the cytochrome b6f complex. During its occurrence, there is also a strong imbalance in the activities of the two photosystems for linear electron flow, in favor of PS II. These experiments may thus serve to establish an in vitro model system for a future investigation of effects related to changes in the imbalance between the two photosystems and its regulation. Furthermore, this experimental system may also be utilized to study the role of the internal lumenal pH in control of photosynthesis.     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

A lesion-mimic mutant of rice (cv. Sekiguchi-asahi) showed enhanced resistance to Magnaporthe grisea infection, thereby inducing Sekiguchi lesion ( sl ) formation and tryptamine accumulation under light. Both Sekiguchi lesion formation and tryptamine accumulation in leaves infected with M. grisea were inhibited by pretreatment with the photosynthetic inhibitor, 3-(3, 4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which suppressed the gene expression of tryptophan decarboxylase ( TDC ), monoamine oxidase activity, H₂O₂ generation and DNA fragmentation. Catalase activity was inhibited by M. grisea infection under light, but magnitude of the inhibition was reduced in leaves pretreated with DCMU. Furthermore, tryptophan accumulated in M. grisea- infected leaves under light but not in DCMU-pretreated ones. Interestingly, such DCMU inhibition was reduced in the presence of tryptophan. Our studies suggest that chloroplasts function as the inhibitor of anti-oxidant system such as catalase activity and the supplier of a precursor of tryptamine and tryptophan in the sl mutant infected with M. grisea .     

Nitrogen fixation by Anabaena cylindrica

Summary The effects of oxygen, light and photosynthesis inhibitors on nitrogenase activities in Anabaena cylindrica batch cultures were followed as a function of time after inoculation. During the early rapid growth period the nitrogenase activities of cultures grown under air/CO₂ or N₂/CO₂ were relatively resistant to oxygen and DCMU inhibition. These cultures also exhibited oxygen-dependent nitrogenase activity in the dark of up to 50% of that measured in the light. After active growth ceased the cultures continued to slowly grow for a prolonged period of time. The nitrogenase activities of these old cultures were very sensitive to oxygen and DCMU inhibition. These cultures also had little or no dark nitrogenase activities. The photosynthesis inhibitor DBMIB was not a specific inhibitor of light-driven electron transport since it inhibited both light and dark nitrogenase activities. Nitrogenase activities induced under oxygen-free/CO₂ gas mixtures initially were significantly more sensitive to oxygen inhibition than those induced under air/CO₂. We discuss these results in relation to heterocyst function.     

Differential effects of DBMIB and DNP-INT on the kinetic phases of P-700 reduction

In the presence of ferredoxin and NADP, DBMIB abolishes the fast-relaxing portion of P-700 together with the reduction of NADP. The slow-relaxing portion is inhibited at much higher concentrations. Qualitatively similar results have been observed with DNP-INT. However, its action appears to be a light-dependent process. The slow, cyclic turnover of P-700 in the presence of DCMU, ferredoxin and NADPH is inhibited by DBMIB but only slightly by DNP-INT. The data suggest that the inhibitors act at different sites of the electron-transport system.