Diaminobenzidine an electron donor to photosystem 1 and to photosystem 2 in chloroplasts.

1. 3,3'-Diaminobenzidine was shown to serve as an electron donor to photosystem 1 in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In Tris-treated chloroplasts diaminobenzidine serves as an electron donor to photosystem 1 and to photosystem 2; the latter is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 2. Addition of diaminobenzidine to Tris-treated chloroplasts causes an increase in fluorescence yield. 3. Diaminobenzidine-dependent electron transport mediated by photosystem 2 is coupled to synthesis of ATP even in the absence of an electron acceptor. This phosphorylation which is presumably supported by cyclic electron flow, is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 4. Diaminobenzidine-dependent ATP formation, in Tris-treated chloroplasts exhibits the red-drop phenomenon. 5. The diaminobenzidine-induced cyclic photophosphorylation (mediated by photosystem 2) is resistant to a large extent to KCN-treatment which is known to inhibit reactions catalyzed by photosystem 1. On the other hand ATP formation supported by electron transport from diaminobenzidine to methyl viologen [in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea] is largely inhibited by KCN-treatment. This observation suggests that there are two coupling sites of ATP formation, one catalyzed by diaminobenzidine as a donor to photosystem 1 (in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and the other supported by diaminobenzidine which acts both as a donor to photosystem 2 (in Tris-treated chloroplasts) and as an acceptor (in its oxidized form) from a carrier located between the two photosystems.     

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-Q_A-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, K_i, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the Q_B niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.Abbreviations BSA bovine seroalbumin - Chl chlorophyll - DCBQ 2,6-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2-(N-morpholino)-ethanesulphonic acid - Pheo pheophytin - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - PS Photosystem - RC reaction center - Tricine N-[Tris(hydroxymethyl)-methyl]-glycine     

On the nature of the photosynthetic energy storage monitored by photoacoustic spectroscopy

The photosynthetic energy storage yield of uncoupled thylakoid membranes was monitored by photoacoustic spectroscopy at various measuring beam intensities. The energy storage rate as evaluated by the half-saturation measuring beam intensity (i₅₀) was inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea, by heat inactivation or by artificial electron acceptors specific for photosystem I or photosystem II; and was activated by electron donors to photosystem I. The reactions involving both photosystems were all characterized by a similar maximal energy storage yield of 16±2 percent. The data could be interpreted if we assumed that the energy storage elicited by the photosystems at 35 Hz is detected at the level of the plastoquinone pool.Abbreviations PS photosystem - Tes N-Tris [hydroxymethl] methyl-2-aminoethanesulfonic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - FeCN potassium ferricyanide - DCBQ 2,5-dichlorobenzoquinone - TMPD N,N,N-tetramethyl-p-phenilenediamine     

Signaling mechanism mediating the anti-senescence effect of low-concentration chemical stressors sprayed onto bean seedlings

The anti-senescence effect of low-concentrations of some stress-inducing compounds of different toxicity and chemical nature such as Cd and Pb salts or 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was investigated in the primary leaves of bean seedlings. In these leaves treated by spraying, chlorophyll content and photosynthetic activity increased, the active cytokinin content of roots and leaves was also significantly higher as compared to the control. These symptoms of stimulation could be eliminated by the inhibitors Li and PD [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one] added to each agent, indicating the involvement of PIP₂-IP₃/DAG and MAPK signaling pathways, respectively, in the response.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Nitrous oxide production by cyanobacteria

Evidence is presented here that axenic cultures of Nostoc spp., Aphanocapsa (PCC 6308), and Aphanocapsa (PCC 6714) but not Anacystis nidulans R-2 (PCC 7942) produce N₂O and ammonia when grown on nitrite. The data suggest that the cyanobacteria produce N₂O by nitrite reduction to ammonia.Nonstandard abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - NIR nitrite reductase     

Loss of sulfide adaptation ability in a thermophilic Oscillatoria

A spontaneous variant incapable of anoxygenic photosynthesis was derived from a fully competent strain of Oscillatoria amphigramulata which was originally isolated from a high sulfide-containing hot spring of New Zealand. Although the variant (Oa-2) acquired a slight ability to photosynthesize in the presence of 0.3–0.4 mM sulfide, this was only after a 24 h exposure to sulfide and represented oxygenic photosynthesis only. Unlike the parent strain, the incompetent variant never grew in the presence of sulfide >0.05 mM, nor was there any relief of the inhibition by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of CO₂ photoincorporation when sulfide was present. The variant strain has retained all of these characteristics over a 4 year period with monthyl transfers in non-sulfide medium. The wild type, under identical conditions, has retained all of its competence with respect to sulfide.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Stimulation of photosystem I-induced oxidation of chloroplast cytochrome b-559 by pre-illumination and by low pH.

(1) The proportion of higher plant chloroplast cytochrome b-559 oxidizable during illumination by low intensity 732 nm light increases as the pH is decreased below 6.5. At pH 5.0-5.3 total oxidation is seen and subsequent red light can cause reduction of up to 2/3 of the oxidized cytochrome. The oxidation by far red light at pH 5 is inhibited by 2 muM 2,5-dibromo-3-methyl-6-isopropyl-rho-benzoquinone whereas the red light-induced reduction is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In this pH range ferricyanide-oxidized cytochrome b-559 exists in a form not reducible by ferrocyanide. (2) An increase in the amplitude of far-red induced oxidation also occurs at higher pH (up to pH 7.8) after pre-treatment of chloroplasts with substantially higher levels of light (approx. 10(6) ergs-cm-2-s-1). The degree of light activation is pH dependent, being more pronounced at lower pH. After light activation, cytochrome b-559 can be completely oxidized by far-red light in a manner reversible by red light up to pH values of 6, and the curve describing the amplitude of far-red oxidation as a function of pH is shifted by 0.5-1.0 pH unit toward higher pH. Far-red oxidation and red light reduction are again inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, respectively. (3) Light activation at pH 5.2-6.0 is also manifested in a small decrease in the amplitude of subsequent dark ferrocyanide reduction, and this decrease is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (10 muM). (4) The effect of intramembranal acidity on the effective redox potential of cytochrome b-559 and its function is discussed.     

Effects of gassing,pH, quenching agents and photodynamically active compounds on photobleaching and photoinhibition of the cyanobacterium Anabaena variabilis

The findings presented in this paper support the suggestion that in the cyanobacterium Anabaena variabilis photobleaching is the result of an increased intracellular level of singlet molecular oxygen, whereas photoinhibition is controlled by a different molecular mechanism. Photobleaching of Anabaena trichomes can be prevented effectively by gassing with argon, nitrogen and carbon dioxide as well as by treatment with the ¹O₂ quenchers sodium azide and crocetin, and finally, with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). On the other hand, photodynamically active compounds, capable of ¹O₂ generation, increase photobleaching drastically. Thus, photobleaching is probably caused by singlet molecular oxygen. Photoinhibition studied with the aid of the fluorescence induction was not prevented by most of the treatments which prevent photobleaching. Therefore, different control mechanisms have to be assumed for this process.Abbreviations DABCO 1,4-diazabicyclo(2,2,2)octane - DBMIB dibromothymoquinone = (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - C-PC C-phycocyanin - Chl a chlorophyll a - LFE low fluence rate exposure - HFE high fluence rate exposure     

Light-induced accumulation of lactate and succinate in Anabaena cylindrica

In the cyanobacterium Anabaena cylindrica lactate accumulated in large amounts when the cells were exposed to light. The presence or absence of oxygen, or a change in CO₂ concentration did not affect the lactate accumulation. The cellular succinate level also increased in the light when CO₂ was supplied at the high concentration of 1%. 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), an inhibitor of photosynthetic electron flow, inhibited the increase in the concentration of lactate and succinate. Photosynthesis is a prerequisite for the increase of these organic acids. Thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase, inhibited the increase of succinate, suggesting that the succinate is formed via fumarate by the reverse of reactions of tricarboxylic acid (TCA) cycle. Upon addition of ammonium to the cell suspension in the light under high CO₂ concentration, the increases in the concentrations of lactate and succinate were inhibited while those of glutamine, glutamate and aspartate were stimulated. Ammonium apparently changed the products of metabolism of pyruvate and oxaloacetate from lactate and succinate to amino acids.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - TTFA thenoyltrifluoroacetone - PCA perchloric acid     

Regulation of ribulose diphosphate formation in vivo by light

Light-dependent formation of ribulose-1,5 diphosphate is completely inhibited by low concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which do not severely affect cyclic photophosphorylation. Also in Scenedesmus mutant number 11, capable of cyclic photophosphorylation, cellular ribulose-1,5 diphosphate-levels do not increase upon illumination. When mutant cells are H₂ adapted, however, a light-dependent formation of ribulose-1,5 diphosphate is observed in the presence of H₂. From these results it has been concluded that at least part of the Calvin cycle does not operate in the dark, since a reductant is lacking which is generated in the light.     

Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (> 5 mg/ml)) is associated with Photosystem I, since it supports phosphorylation with a P/e₂ value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e₂ value routinely observed is about 0.2. Bovine serum albumin (1–2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even in the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Photosynthetic electron transfer and membrane energization in spheroplasts and membrane vesicles of the thermophilic cyanobacterium Synechoccus 6716

The higher the incubation temperature, the higher the light intensity that membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 require for the saturation of O₂-production. If membrane vesicles are incubated at temperatures at which intact cells are growing optimally, photosynthetic O₂-production and membrane energization decrease rapidly, suggesting that the thermophilic properties are rapidly lost. If membrane integrity is maintained (spheroplasts) the harmful effect of higher temperatures is much less. The effects of 2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone (DBMIB), 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide (S-13), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and N,N-dicyclohexylcarbodiimide (DCCD) are the same as in chloroplasts, be it that DCCD acts as an electron transfer inhibitor at higher concentrations. The supposed alternative site of DCMU inhibition in cyanobacteria is rejected.Spheroplasts show a reversible energy-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) caused by illumination. ATP hydrolysis only give rise to fluorescence quenching in membrane vesicles. Long incubation at higher temperatures reduces the fluorescence quenching of membrane vesicles and spheroplasts, the latter being more stable than the former.Abbreviations 9AA 9-aminoacridine - ACMA 9-amino-6-chloro-2-methoxyacridine - Chl chlorophyll - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DCP 1,5-diphenylcarbazide - PMS methyl-phenazoniummethosulfate - PS-I photosystem I - PS-II photosystem II - S-13 5-chloro-3-t-butyl-2 chloro-4-nitrosalicylanilide     

Light versus Dark Carbon Metabolism in Cherry Tomato Fruits: I. Occurrence of Photosynthesis. Study of the Intermediates

The photosynthetic properties of the internal and peripheral tissues of the cherry tomato fruit (Lycopersicum esculentum var. cerasiforme Dun A. Gray) were investigated. Whole fruit and their isolated tissues evolve large amounts of CO₂ in darkness. In the light, this evolution decreases but nevertheless remains a net evolution; 3-(3,4-dichlorophenyl)-1,1-dimethylurea abolishes the effects of light.     

Visualization of a rapid,red/far-red light-dependent reaction by centrifuge microscopy

Summary Using a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.Abbreviations APW artificial pond water - CMS centrifuge microscope of the stroboscopic type - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr phytochrome, far-red light-absorbing form - Pr phytochrome, red light-absorbing form     

Electron Transport-Dependent Chlorophyll-a Fluorescence Quenching by O(2) in Various Algae and Higher Plants

A comparison of chlorophyll-a fluorescence in brown algae (Macrocystis integrifolia, Fucus vesiculosis), green algae (Scenedesmus obliquus, Ulva sp.) and higher plants (bean, corn) show differences in the relative fluorescence intensities and induction time courses which characterize each type of plant. These differences are not reflected in either the maximum fluorescence emission in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (F_max) or the nonvariable fluorescence (F_o). Constancy of F_o and F_max suggests functional similarities of photosystem II and associated antennae pigments in the various classes of plants. The time course differences are observed only in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and appear, therefore, to be electron transport dependent. During induction, the peak in fluorescence (F_p) is much lower in all of the algae studied than in the higher plants. Exogenous O₂ strongly quenches F_p in all plants studied and our data indicate that the low F_p in the algae can be partially accounted for by endogenous O₂ quenching.     

Effect of oxygen on the cyanobacterium Oscillatoria limnetica

Oscillatoria limnetica grown photoautotrophically under aerobic or anaerobic conditions contained a single superoxide dismutase (SOD) of identical electrophoretic mobility in both cases. Its activity was cyanide resistant and H₂O₂ sensitive, implicating Fe-SOD. The enzyme level was high in aerobically and low in anaerobically growing cells. Anaerobically grown cells were more sensitive than aerobic to photooxidation, as expressed by bleaching of phycocyanin and disintegration of the trichomes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - SOD superoxide dismutase     

Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii

A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm.     

Synthesis and bronchospasmolytic properties of some pyridazinone derivatives

The synthesis and evaluaton of the biological activity of a series of 3(2H)-pyridazinones is reported. The bronchodilatating activity of these compounds was determined on the guinea pig trachea. Compounds 6 and 17 with 1-(4-amino-3,5-dichlorophenyl)-2-hydroxyethyl-and 1-(3,4-dichlorophenyl)-2-hydroxyethyl groups linked through a piperazine ring to the 6-position of 3(2H)-pyridazinone show a good bronchospasmolytic activity.