μM DCMU Research Articles

Photophobotaxis in the filamentous cyanobacterium Phormidium lacuna: Mechanisms and implications for photosynthesis-based light direction sensing.

Cyanobacterium Phormidium lacuna filaments move from dark to illuminated areas by twitching motility. Time-lapse recordings demonstrated that this photophobotaxis response was based on random movements with movement reversion at the light-dark border. The filaments in the illuminated area form a biofilm attached to the surface. The wild-type and the pixJ and cphA mutants were investigated for photophobotaxis at diverse wavelengths and intensities. CphA is a cyanobacterial phytochrome; PixJ is a biliprotein with a methyl-accepting chemotaxis domain and is regarded as a phototaxis photoreceptor in other species. The cphA mutant exhibited reduced biofilm surface binding. The pixJ mutant was characterized as a negative photophobotaxis regulator and not as a light direction sensor. 3-(3,4-dichlorophenyl)1,1-dimethylurea (DCMU) blocks electron transfer in PS II. At concentrations of 100 and 1000 μM DCMU, photophobotaxis was inhibited to a greater extent than motility, suggesting that PSII has a role in photophobotaxis. We argue that the intracellular concentrations of regular photoreceptors, including CphA or PixJ, are too small for a filament to sense rapid light intensity changes in very weak light. Three arguments, specific inhibition by DCMU, broad spectral sensitivity, and sensitivity against weak light, support photosynthesis pigments for use as photophobotaxis sensors.

New cyanobacterial strains for biohydrogen production

Under certain conditions, cyanobacteria can switch from photosynthesis to hydrogen production, which is a good energy carrier. However, the biological diversity of hydrogen-releasing cyanobacteria has a great unexplored potential. This study is aimed to investigate the ability of new strains of cyanobacteria Cyanobacterium sp. IPPAS B-1200, Dolichospermum sp. IPPAS B-1213, and Sodalinema gerasimenkoae IPPAS B-353 to release H2 and to evaluate the effects of photosystem II inhibitor 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) on H2 production under light and dark conditions. The results showed that cultures treated with DCMU produced several times more H2 than untreated cells. The highest rate of H2 photoproduction of 4.24 μmol H2 (mg Chl a h)−1 was found in a Dolichospermum sp. IPPAS B-1213 culture treated with 20 μM DCMU.

Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is a potential dark fermentative producer of molecular hydrogen (H2) that produces very little H2 under illumination. One factor limiting the H2 photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O2) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H2 production of A. halophytica under light and dark conditions and on photosynthetic and respiratory activities. The results showed that A. halophytica treated with CCCP and DCMU produced H2 at three to five times the rate of untreated cells, when exposed to light. The highest H2 photoproduction rates, 2.26 ± 0.24 and 3.63 ± 0.26 μmol H2 g−1 dry weight h−1, were found in cells treated with 0.5 μM CCCP and 50 μM DCMU, respectively. Without inhibitor treatment, A. halophytica incubated in the dark showed a significant increase in H2 production compared with cells that were incubated in the light. Only CCCP treatment increased H2 production of A. halophytica during dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H2 production rate of 39.50 ± 2.13 μmol H2 g−1 dry weight h−1 was found in cells treated with 0.5 μM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O2, which promoted bidirectional hydrogenase activity in A. halophytica cells. In addition, only CCCP enhanced the respiration rate, further reducing the O2 level. In contrast, DCMU reduced the respiration rate in A. halophytica.

Acceleration of cyclic electron flow in rice plants (Oryza sativa L.) deficient in the PsbS protein of Photosystem II

When compared with Photosystem I (PSI) in wild-type (WT) rice plants, PSI in PsbS-knockout (KO) plants that lack the energy-dependent component of nonphotochemical quenching (NPQ) was less sensitive to photoinhibition. Therefore, we investigated the relationship between NPQ and cyclic electron flow (CEF) around PSI as a photoprotective mechanism. Activities of two CEF routes (PGR5-dependent or NDH-dependent) were compared between those genotypes by using both dark-adapted plants and pre-illuminated plants, i.e., those in which the Calvin–Benson cycle is de-activated and activated, respectively. In dark-adapted leaves activity of the PGR5-dependent route was determined as the rate of P700 photooxidation. Activity was higher in the mutants than in the WT. However, no difference was noted when plants of either genotype were pre-illuminated. When the electron transport pathway was switched to the cyclic mode by infiltrating leaf segments with 150 mM sorbitol, 40 μM DCMU, and 2 mM hydroxylamine, the rate of P700 oxidation was faster in the mutant. That difference disappeared when leaves were infiltrated with antimycin A to inhibit the PGR5-dependent route. Chlorophyll fluorescence (Fo) was also evaluated. To achieve an Fo level comparable to that of the WT, activation of the NDH-dependent route in the mutant required pre-illumination at a certain dose. Therefore, we propose that, as an alternate pathway for the photoprotection of photosystems in the absence of energy-dependent quenching, this PGR5-dependent route is more highly activated in the PsbS-KO mutants than in the WT. Moreover, that stronger activity is probably responsible for slower activation of the NDH-dependent route in the mutant.

Improving conversion efficiency of solar energy to electricity in cyanobacterial PEMFC by high levels of photo-H2 production

In this study, we described an efficient electrical power generating system containing cyanobacterial photo-H2 production and custom-built proton exchange membrane fuel cell (PEMFC). The filamentous N2-fixing cyanobacterium Anabaena cylindrica was used as the photo-H2 producer. A photosynthesis inhibitor-diuron (DCMU) was used for the enhancement of photo-H2 production in the culture under argon gas. For the first time, a total of 1.0 μM DCMU was found to be the most effective treatment, as this produced 3.6 fold higher levels of H2 in microalgae. By measuring polarization curve, the gas mixture collected from the culture was proven to be an effective fuel for electrical generation through a custom-built PEMFC. When the PEMFC was directly combined with the culture tube, the cells generated as much as 843 mV during a 5-day incubation due to the efficient conversion of solar energy to H2 by A. cylindrica. Light energy conversion efficiency (LCE) for both solar energy to H2 and solar energy to electricity were also determined. The LCE for the cyanobacterial conversion of solar energy to H2 reached a peak at four days with a maximal value of 2.05% and an average value of 1.70% ± 0.17. The corresponding LCE for the conversion of solar energy to electricity in this system was 1.13% at peak and 0.94% ± 0.09 on average.

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.

Mutation of algae from sensitivity to resistance against environmental selective agents: the ecological genetics of Dictyosphaerium chlorelloides (Chlorophyceae) under lethal doses of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea herbicide

Residues contribute to water pollution and pose a challenge to microalgal populations because numerous contaminants are toxic to microalgae, even in the micromolar range. Adaptation of microalgae from herbicide sensitivity to herbicide resistance was analysed by an ecological genetic approach, using the unicellular alga Dictyosphaerium chlorelloides (Chlorophyceae) as an experimental model. A dose–effect study showed that the Malthusian parameter under conditions of r selection in an uncrowded environment and the carrying capacity under conditions of K selection in an crowded environment were both restricted even by low concentrations (< 1 μM) of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) herbicide. When a culture was treated with 50 μM DCMU, it cleared after a few days, as a result of destruction of sensitive cells by the herbicide. However, after further incubation for several days, the culture sometimes regained colour, owing to the growth of cell variants resistant to the herbicide. A fluctuation analysis was carried out to distinguish between (1) herbicide-resistant cells arising by direct and specific acquired adaptation in response to the herbicide and (2) herbicide-resistant cells arising by rare spontaneous mutations occurring randomly during replication of organisms prior to the incorporation of the herbicide. The fluctuation analysis unequivocally demonstrated that DCMU is not facilitating the development of DCMU-resistant cells; rather, we found that DCMU-resistant cells occur spontaneously by mutation in nonselective conditions prior to the incorporation of the herbicide (preselective mutations). The rate of spontaneous mutation from DCMU sensitivity to DCMU resistance was ∼ 2.2 × 10−6 mutants per cell division. Mutation was recurrent from a normal wild-type DCMU-sensitive allele to an DCMU-resistant allele, but such herbicide-resistant alleles were detrimental in terms of fitness in the absence of the herbicide. A competition experiment between wild-type DCMU-sensitive cells and DCMU-resistant mutants showed that, in small populations, the DCMU-resistant mutants are driven to extinction. The resistant variants are maintained in the absence of the herbicide as the result of a balance between new resistant cells arising by rare spontaneous mutation and resistant cells eliminated by natural selection. In our case, the average frequency of DCMU-resistant mutants in the absence of the DCMU is about five DCMU-resistant mutants per million cells. The results of our experimental model suggest that spontaneous mutation from herbicide sensitivity to herbicide resistance is sufficient in itself to assure the survival of microalgae populations in herbicide-contaminated environments when the population size is large.

Energy storage and optical cross-section of PS I in the cyanobacterium Synechococcus PCC 7002 and a psaE- mutant

In vivo electron flow in the unicellular cyanobacterium Synechococcus sp. PCC 7002 was studied by pulsed, time- resolved photoacoustics (PTRPA). Using 1-μs, 2 μJ or 1013 hνcm-2 pulses at 695 nm, we observed large (42 ± 2%) Photosystem I (PS I) cyclic energy storage (ES) in the period of 2 to 12 ms after excitation with wild type (WT) intact cells. This cyclic ES was insensitive to flash interval from 0.3 to 10 s and to the presence of 1 μm DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). At this low flash energy and in the absence of continuous background light (in the dark), antimycin A, carbonylcyanide-m- chlorophenylhydrazone (CCCP), 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), DCMU, 2- n-heptyl-4- hydroxyquinoline-N-oxide (HQNO), myxothiazol and N- ethylmaleimide (NEM) caused little or no inhibition of PS I cyclic electron flow. When continuous background far-red light (λ > 715 nm) was added during the measurement, strong inhibition by DBMIB and NEM and less by HQNO was observed, the amplitude of which was related to both concentration and the intensity of the background light. Analysis of the data with DBMIB yields its binding constant, 1 μm, and the turnover time of the system (> 20 ms). A turnover time of the uninhibited system of 2–3 ms was obtained by a pump-probe method. A dramatic lifting of the partial inhibition in the presence of far-red light was caused by antimycin A and a smaller effect by myxothiazol. The rescuing effect was assigned to a ‘short circuiting’ of the electron flow about the cytochrome (cyt) b6/f system. Progressively increasing the laser pulse energy allowed us to calculate the PS I optical cross-section (54 ± 2 A). Analysis by the sensitive method of convolutions revealed a possible energy loss on the few ms time scale by antimycin A in the dark. The analysis also revealed a similar effect or artifact in uninhibited samples using the same sample illuminated with saturating continuous light, the standard procedure in photoacoustics (PA). A psaE- mutant showed more inhibition in the dark by DBMIB and with far-red light by HQNO, but less inhibition in the far-red light by myxothiazol than the WT. Under normal growth conditions, maximum ES for the psaE- mutant (38 ± 2%) was similar to that of the WT (42 ± 2%). However, under mild heat stress, maximum ES for the psaE- mutant dropped to 26% while the WT maximum ES stayed unchanged at 41%, within batch-to-batch variation. These results would indicate that the PsaE protein is not essential for PS I cyclic electron flow under our experimental conditions but plays a stabilizing role in the PS I complex under a mild thermal stress.

An assessment of the cage-culture turbidostat as an alternative algal bioassay

A computer controlled cage-culture turbidostat (CCT) is described as an alternative to batch algal bioassays. The CCT is designed to monitor algal growth changes in response to the addition of toxicants including chronic toxic effects in comparison to batch culture toxicity tests which are most successful in assessing acute responses. The system is easily and quickly set up, with minimum operator time required once assembled. The alga Phaeodactylum tricornutum (CCAP 1052/6) was used as the test species. Stable growth of the alga could be maintained in the CCT for 14 days, sufficient time to run bioassay experiments using both the herbicide Diuron ™ (DCMU) and a complex industrial effluent. Additions of various concentrations of both toxins to the chamber caused changes in the growth rate and the chl- a concentration of the culture. The Median Inhibitory Concentration of effluent calculated to cause a 50% reduction in the growth rate (IC 50) in the CCT was estimated to be 0.33 μM DCMU, and 15.33% of the effluent. Comparable results were obtained when using the standard algal growth inhibition toxicity (AGIT) test, the 72 h-IC 50 values were 0.09 μM DCMU and 16.4% effluent. This study indicates a potential use for the CCT for water quality monitoring, which may provide more environmentally relevant information than the commonly used AGIT test.

Function of plastoquinones B and C as electron acceptors in Photosystem II and fatty acid analysis of plastoquinone B

We have found that in petroleum-ether extracted tobacco thylakoids, plastoquinone A (PQ-A) and plastoquinone C (PQ-C) had similar efficiency in restoration of oxygen-evolving activity, while plastoquinone B (PQ-B), which is a fatty acid ester of PQ-C, was about 50% less effective. This indicates that apart from PQ-A, PQ-C and to a smaller extent PQ-B may function as electron acceptors of Photosystem II (PS II). The DCMU inhibition curves for PQ-C and PQ-B were biphasic and an initial slow decline was followed by a sharp decrease in oxygen evolution yield with a 50% inhibition (I50) at 0.25 μM DCMU. In the case of PQ-A (I50 = 0.20 μM DCMU), the activity decreased gradually without the sharp transition. The corresponding inhibition curve for unextracted thylakoids, where all the native prenylquinones are present, shows an intermediate shape between PQ-A and PQ-C but with a higher I50, equal to 0.32 μM, suggesting that the contribution of PQ-C as an electron acceptor of Photosystem II might be significant in thylakoid membranes with natural prenyllipid composition. α-Tocopherol quinone showed no activity in the restoration of oxygen evolution in extracted thylakoids, indicating that it cannot accept electrons from PS II. The fatty acid composition of PQ-B isolated from maple leaves showed a high degree of saturated fatty acids like myristic and palmitic acid, and its unique composition indicates that it is a natural component of the thylakoid membrane.

A mutant of the red microalga Porphyridium sp. (Rhodophyceae) resistant to DCMU and atrazine

A mutant (DC-2) of the unicellular red alga Porphyridium sp. (UTEX 637) resistant to the herbicide diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU] was isolated after induced mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NNG) at a frequency of 2.1 × 10−5. The minimal concentration of DCMU that completely inhibited growth of the wild type was fiftyfold lower than that required for complete inhibition of the mutant's growth. The mutant was found to be stable over several consecutive growth cycles in the absence of DCMU. When the wild type was exposed to a sub-lethal concentration of DCMU (0.2 μM),its photosynthesis was reduced by up to 72%, while the mutant was not affected by this concentration. In the presence of up to 5.0 μM DCMU the Pmax of the mutant was slightly reduced. However, these concentrations completely inhibited photosynthesis in the wild type. DCMU at a concentration of 0.2 μM decreased the cellular level of phycobiliproteins (phycoerythrin and phycocyanin) but not of chlorophyll a, while no change was recorded in the level of these pigments in the mutant culture grown with 2.5 μM DCMU. Sulphated polysaccharides responded in the opposite way to phycobiliproteins: their total content did not change at 0.2 μM DCMU in the wild type, while that of DC-2 was reduced by 38%. DC-2 mutant proved to be resistant to another photosystem II-inhibiting herbicide, 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine). It grew in a medium containing up to 100-μM atrazine, whereas growth of the wild type was completely inhibited at 5.0 μM.

Functional characterisation of a purified homogeneous Photosystem II core complex with high oxygen evolution capacity from spinach

The functional properties of a purified homogeneous spinach PS II-core complex with high oxygen evolution capacity (Haag et al. 1990a) were investigated in detail by measuring thermoluminescence and oscillation patterns of flash induced oxygen evolution and fluorescence quantum yield changes. The following results were obtained: a) Depending on the illumination conditions the PS II-core complexes exhibit several thermoluminescence bands corresponding to the A band, Q band and Zv band in PS II membrane fragments. The lifetime of the Q band (Tmax=10°C) was determined to be 8s at T=10°C. No B band corresponding to S2QB (-) or S3QB (-) recombination could be detected. b) The flash induced transient fluorescence quantum yield changes exhibit a multiphasi relaxation kinetics shich reflect the reoxidation of Q A (-) . In control samples without exogenous acceptors this process is markedly slower than in PS II membrane fragments. The reaction becomes significantly retarded by addition of 10 μM DCMU. After dark incubation in the presence of K3[Fe(CN)6 c) Excitation of dark-adapted samples with a train of short saturating flashes gives rise to a typical pattern dominated by a high O2 yield due to the third flash and a highly damped period four oscillation. The decay of redox states S2 and S3 are dominated by short life times of 4.3 s and 1.5 s, respectively, at 20°C. The results of the present study reveal that in purified homogeneous PS II-core complexes with high oxygen evolution isolated from higher plants by β-dodecylmaltoside solubilization the thermodynamic properties and the kinetic parameters of the redox groups leading to electron transfer from water to QA are well preserved. The most obvious phenomenon is a severe modification of the QB binding site. The implications of this finding are discussed.

Two types of PS II centres as manifested by light saturation of delayed fluorescence from DCMU-treated chloroplasts

Intensity of 2 s delayed fluorescence (DF) as a function of steady-state actinic light intensity was investigated in pea chloroplasts in the presence of 10 μM DCMU. The light saturation curve of DF was approximated by a sum of two hyperbolic components which differ by an order of magnitude in the half-saturating incident light intensity. The relative contribution of the amplitudes of the components was practically independent of cation (Na(+) and Mg(2+)) concentration and a short-term heating of the chloroplasts at 45°C. The component saturating at low incident light intensity was selectively suppressed by 100 μM DCMU or by 1 μmol μg(-1) Chl oleic acid. DF intensity following excitation by a single saturating 15 μs flash was equal to the intensity of the component saturating at a low incident light intensity. Upon flash excitation, the maximum steady-state DF level was found to be attained only after a series of saturating flashes. It is concluded that the two components of the DF light saturation curves are related to PS II centres heterogeneity in quantum yield of stabilization of the reduced primary quinone acceptor.

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl- p-benzoquinone (Ph- p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q B-site of Ph- p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q A (possibly also at the Q B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.

Electron transfer through the quinone acceptor complex of Photosystem II after one or two actinic flashes in bicarbonate-depleted spinach thylakoid membranes

We report here the pH dependence of the kinetics of the decay of variable chlorophyll a fluorescence after one or two actinic flashes in the absence or the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl-urea) in HCO 2 −-depleted or anion-inhibited spinach thylakoid membranes. All the reported effects of HCO 3 − removal are reversed by the addition of 5 mM HCO 3 −. The initial first-order component for the oxidation of Q A − (the reduced primary plastoquinone acceptor of Photosystem II (PS II) by Q B (the secondary plastoquinone acceptor) was reversibly inhibited in a pH-dependent manner in HCO 3 −-depleted membranes. After a single actinic flash, the half-time of Q A − decay was 630 μs (amplitude, 29%) at pH 6.5 which changed to a value of 320 μs (amplitude, 66%) at pH 7.75. The rate and amplitude at pH 7.75 were approximately the same as found in the restored and control membranes which were pH independent over the same pH range. A similar observation was made after the second actinic flash. Thus, at alkaline pH HCO 3 −-depleted membranes behave as control membranes with respect to electron flow from Q A to Q B or to Q B −. The time ( t 50) at which the [Q A −] is 50% of the maximum [Q A −] during the back reaction between Q A − and the S 2 state of the oxygen-evolving complex, in the presence of 5 μM DCMU, was increased from 1.3 s in control and restored samples to 5.3 s in HCO 3 −-depleted samples below pH 7.0, but was unaffected above pH 7.5 (2.3–2.9 s in all cases). Furthermore, a new pathway of Q A − with a half-time of less than 100 μs was present at pH 8.0 in the presence of DCMU, in approx. one-third of the PS II centers in HCO 3 −-depleted membranes. The apparent equilibrium for the sharing of an electron between Q A and Q B is estimated to decrease by a factor of 4 at pH 6.0 in treated membranes ( K app ≈ 16) as compared to the restored or control membranes ( K app ≈ 62); there was no difference in K app at pH 7.75. Estimates of the operating redox potential for the Q B/Q B − couple from the results presented here indicated that the pH dependence of this parameter is greatly reduced in treated membranes (−60 mV at pH 6.0 to −72 mV at pH 7.75) as compared to restored or control membranes (−25 mV at pH 6.0 to −72 mV at pH 7.75). We iscuss our results in the context of a model that envisages HCO 3 − to act as a proton donor to the protein dissociable group believed to participate in the protonation of Q B −. Finally, the possibility of HCO 3 − being a ligand to Fe 2+ in the Q A-Fe-Q B complex of the PS II reaction center is also discussed.

Assessment of the effects of the photosynthesis-inhibiting herbicides Diuron, DCMU, metamitron and metribuzin on growth and nitrogenase activity of Nostoc muscorum and a new cyanobacterial isolate, strain G4

The influence of the photosynthesis-inhibiting herbicides Diuron, DCMU, metamitron, and metribuzin on growth and nitrogenase activity of Nostoc muscorum and a new cyanobacterial isolate, strain G4, was studied. The experiments were performed under N2-fixing photoautotrophic conditions. Both cyanobacteria showed a high degree of tolerance towards the herbicides tested. In the presence of metamitron (35, 70, and 140 ppm) and metribuzin (7, 14, 35, and 70 ppm) strain G4 proliferated as well as the control culture. Metamitron and metribuzin had no influence on the nitrogenase synthesis of strain G4 and N. muscorum. When treated with 1 μM DCMU, strain G4 and N. muscorum showed partial inhibition for the first few days, but entirely recovered during succeeding incubation. In the presence of 10 ppm Diuron, N. muscorum and strain G4, compared to the control, showed 30% and 80% nitrogenase activity, respectively.

Multiple action sites for photosystem II herbicides as revealed by delayed fluorescence

DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) at concentrations higher than 10 μM suppresses the second time range delayed fluorescence (DF) of pea chloroplasts, due to inhibition of the oxidizing side of photosystem II (PS II). The inhibition of the reducing side of PS II resulting in the suppression of millisecond DF takes place at much lower (∼0.01 μM) DCMU concentrations. The variation in the herbicide-affinities of the reducing and oxidizing sides of PS II is not the same for DCMU and phenol-type herbicides. The DCMU-affinity of the oxidizing side considerably increases and approximates that of the reducing side upon mild treatment of chloroplasts with oleic acid. Probably this is a result of some changes in the environment of the binding site at the oxidizing side. At DCMU concentrations higher than 1 mM, the chaotropic action of DCMU leads to the generation of millisecond luminescence which is not related to the functioning of the reaction centres.

Some Factors Controlling the Distribution of Two Pond‐Dwelling Ciliates with Algal Symbionts (Frontonia vernalis and Euplotes daidaleos)1

ABSTRACTThe vertical distributions of two pond‐dwelling zoochlorellae‐bearing ciliates (Euplotes daidaleos Diller &amp; Kounaris, 1966 and Frontonia vernalis Ehrenberg, 1838) were monitored over a 24‐h period. Both species maintained peak abundance at a low O2 level (usually &lt; 1 mg/liter). They did not migrate in response to the changing light level. Experiments with laboratory cultures indicated that the characteristic distribution in an O2 gradient in the dark was largely controlled by the oxygen tension. The increased motility in anoxia and high pO2 was independent of large changes in pCO2 and pH. Ciliates living in anoxia or a very low pO2 would migrate out of the dark and into the dimly lit (10 μE m‐2 sec‐1) part of a glass cell because there they could photosynthesize, produce O2, and create a suitable oxygenated microenvironment; a further increase in the light level caused a slow migration out of the light. Similar migrations were observed when the light level remained low but the pO2 was artificially raised. Ciliates suspended in 1 μM DCMU (an inhibitor of photosynthetic O2 evolution) took longer to migrate into the light and they did not avoid high light levels (&gt; 100, μE m‐2 sec‐1)‐ Frontonia suspended in water with a pO2 of 1% aggregated at a low light level (1 μE m‐2 sec‐1); peak daytime abundance in the pond occurred at about this light level. Frontonia vernalis tends to swim vertically upwards (anterior end up) when suspended in anoxic water. This apparent negative geotaxis compensates for the high sedimentation velocity (0.36 mm sec‐1) of this large ciliate and facilitates its aggregation at the metalimnion. The O2 tension appears to be the principal factor controlling the vertical distributions of both species. Occasional, enhanced convection within the metalimnion has a secondary influence. Light influences the vertical profile only if it promotes photosynthesis and increases the intracellular pO2.

Inhibition of a respiratory activity by short saturating flashes in Chlamydomonas: Evidence for a chlororespiration

Excitation with short actinic flashes (2 μs) of oxygenated dark-adapted Chlamydomonas cells deposited on a bare O 2 platinum electrode induces an increase of the amperometric signal after the first two flashes. Mass spectrometer experiments performed in the presence of 18O 2 showed that this signal was not due to the photolysis of water (H 2 16O). The insensitivity of this signal to 10 μM DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), its stimulation by acetate or high O 2 concentration as well as its inhibition by cyanide indicate that these flash-induced changes in O 2 concentration were related to the inhibition of a respiratory process. Because this rather fast inhibition of respiration is insensitive to antimycin A and to salicyl hydroxamic acid, inhibitors of mitochondrial respiration, and because it occurs on a single flash illumination, we conclude that the related respiratory activity takes place inside the chloroplast (chlororespiration) and not in the mitochondria. This interpretation is confirmed by the quite high K m( O 2) of this process (about 23 μM) compared to those measured for the mitochondrial reactions (0.2 μM for the cytochrome oxidase pathway and 5.5 μM for the alternative pathway). In a mutant lacking Photosystem I activity, no photoinhibition of respiration was observed. We conclude from the above results that the light-induced inhibition of chlororespiration is due to the oxidation by Photosystem I activity of electron carriers common to both photosynthetic and chlororespiratory chains.