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PubMed1979

Comparative study of the fluorescence yield and of the C550 absorption change at room temperature.

The C550 absorption change and the fluorescence yield were studied at room temperature in chloroplasts in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and under conditions in which contributions of P-700 and of the electrochromic effect were neglible. 1. The C550 difference spectrum is a typical band shift with an isobestic point close to 550 nm. 2. The maximum amplitude of C550 absorption change is reached upon the first flash of a series of saturating flashes, unlike the maximum fluorescence yield which is attained after several flashes. 3. The comparison of the induction curves of the C550 change and the fluorescence yield in weak light shows that the fluorescence yield is controlled by two quenchers: one of them (Q1), the redox state of which C550 is a probe, is responsible for the major part of the quenching; the other one (Q2), which is less concentrated and less efficient becomes predominant at the end of the fluorescence induction. 4. Quencher Q2 back-reacts faster than quencher Q1. 5. Two alternative models are discussed in which Q1 and Q2 belong either to the same Photosystem II center or to two different photocenters.

Chloroplasts↗
PubMed1979

Anomalous oxygen uptake from isolated chloroplasts inhibited in photosystem II and without external electron donors.

Light induced modulated signal of oxygen uptake by isolated chloroplasts in the presence of methyl viologen, when Photosystem II activity was inhibited and in the absence of any electron donors, was detected by a modulated oxygen Pt electrode, polarized negatively. Evidence is brought to show that an electrochemical process which takes place on the surface of the negatively polarized Pt-cathode produces an intermediate which serves as an electron donor to Photosystem I. Atempts to identify this intermediate show that it may be very probably the superoxide radical generated by the electrochemical reduction of oxygen which continuously diffuses from the external circulating medium to the electrode.

Cell Membrane↗
PubMed1979

Dichlorophenylurea-resistant oxygen evolution in Chlorella after cerulenin treatment.

Fluorescence spectra at 77 K, oxygen evolution at 30 degrees C and delayed fluorescence at 25 degrees C were measured in Chlorella pyrenoidosa cultures with and without cerulenin and subsequent 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU) treatment, respectively. In pure algal cultures the oxygen evolution was inhibited by DCMU and the long-time component of fluorescence was highly influenced by DCMU, as expected. In contrast, both oxygen evolution and delayed fluorescence became DCMU-resistant in cerulenin-treated cultures. The DCMU-resistance is correlated with a change in the fatty acid distribution of the thylakoid membrane, which also leads to changes in the prompt fluorescence. Cerulenin appears to be a promising new tool of diagnostics for the hitherto unsatisfactorily understood processes of oxygen evolution in photosynthesizing organisms.

Antifungal Agents↗
PubMed1980

The function of diphenylamines as modifiers of photosystem II electron transport in isolated spinach chloroplasts.

Diphenylamines with highly electronegative substituents are effective inhibitors of photosynthetic electron transport and photophosphorylation. They inhibit only Photosystem II- and not Photosystem I-dependent photoreductions. As judged from the missing tetramethylphenylenediamine-bypass, displacement experiments with [14C]metribuzin, and measurements of oxygen evolution in trypsinated chloroplasts, diphenylamines act neither as dibromomethylisopropylbenzoquinone- nor as dichlorophenyldimethylurea-type inhibitors. All of the diphenylamines tested were found to function as ADRY-type reagents, (Renger, G. (1972) Biochim. Biophys, Acta 256, 428-439) which modify the stability of redox equivalents stored within the water-splitting enzyme system Y. The site of inhibition of diphenylamines is assumed to be located at the reducing side of Photosystem II or the reaction center itself. The inhibitory effect could involve a modification of cytochrome b-559 or its surrounding. In an assay for herbicidal activity, diphenylamines showed more pronouncing effect on mono- than on dicotyledonous plants.

Aniline Compounds↗
PubMed1980

The influence of the electric diffusion potential on delayed fluorescence light curves of chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

The potassium salt-induced transient increase of delayed fluorescence yield was studied in pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A simple kinetic model is proposed to account for the actinic light intensity dependence of the delayed fluorescence enhancement by the transmembrane diffusion potential induced by sudden salt addition. The electric field dependence of the rate constants for the recombination of primary separated charges with and without subsequent electronic excitation of reaction center chlorophyll was obtained. From the value of enhancement of delayed fluorescence by salt concentration gradients at saturating actinic light intensity, it is concluded that the distance, normal to thylakoid membrane surface, between the primary acceptor and the donor of Photosystem II is smaller than the membrane thickness.

Chloroplasts↗
PubMed1980

Temperature dependence of the delayed fluorescence from wheat leaves treated with 3-(3,4-dichlorophenyl)-1-1,-dimethylurea.

Light saturation curves of the delayed fluorescence of wheat leaves treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were measured at different temperatures. Calculated activation energies for a half-saturation actinic light intensity and saturated delayed fluorescence emission were 0.89 and 0.32 eV, respectively. On the basis of the kinetic model energy levels of Photosystem II reaction center components were estimated.

Diuron↗
PubMed1980

Electron spin echo studies on chloroplasts. Spectral characteristics of electron transport components and light-induced transients.

Electron-spin resonance echoes are used to study the complex overlapping ESR spectra of whole chloroplasts. By varying the repetition rate of the microwave pulse sequence, delay time, and pulse width, signals with different longitudinal and transverse relaxation times were extracted. We have identified the echo signals due to plastocyanin and ferredoxins. In addition, we have found a strong signal at g = 4.3, that possibly arises from distorted cytochrome, and weak signals in the region g = 6-9. The strong echo signal at g = 2.0047 (Signal II), is made up of at least three "dark" components that differ in their relaxation times. Upon illumination at 1.2 K several of the echo signals including Signal II show reversible light-induced components. The kinetics of these transients depend on the addition of 3(3,4-dichlorophenyl)-1,1-dimethyl urea. Part of the transients are believed to arise from cyclic electron flow around Photosystem I.

Chloroplasts↗
PubMed1980

Acid-induced double hits in glutaraldehyde-treated Chlorella.

Glutaraldehyde-treated Chlorella cells show pH-dependent oxygen evolution characteristics. 1. The oxygen yield on the second flash (Y2) of a sequence of 2 mus flashes is pH-dependent, the rest of the sequence is unchanged. 2. This yield on the second flash increases as the dark-time between sequences is increased. 3. The deactivation times for the S2 and S3 states are not significantly changed at different pH values. 4. Using a short flash (0.5 mus) for the first flash, no oxygen was detected on the second flash, indicating that there is no S2 remaining in the dark. 5. When the intensity of the first flash is varied, a sigmoidal curve is obtained for Y2 as a function of flash intensity of the first flash is varied, a sigmoidal curve is obtained for Y2 as a function of flash intensity at pH 6.0 but not at pH 7.5. This is indicative of a double hit process. 6. At pH 6.0 the turnover of the Photosystem II centers after the first flash has a fast rise (t 1/2 < 25 mus) followed by a slow phase. This behaviour is not seen at pH 7.5, nor after two flashes. 7. We propose a scheme in which an auxiliary acceptor is in series with Q1 and in competition with B. The redox potential of the auxiliary acceptor is pH dependent.

Aldehydes↗
PubMed1980

Energy distribution in the photochemical apparatus of Porphyridium cruentum in state I and state II.

Fluorescence of Porphyridium cruentum in state I (cells equilibrated in light absorbed predominantly by Photosystem I) and in state II (cells equilibrated in light absorbed appreciably by Photosystem II) was examined to determine how the distribution of excitation energy was altered in the transitions between state I and state II. Low temperature emission spectra of cells frozen state I and state II confirmed that a larger fraction of the excitation energy is delivered to Photosystem II in state I. Low temperature measurements showed that the yield of energy transfer from Photosystem II to Photosystem I was greater in state II and calculations indicated that the photochemical rate constant for such energy transfer was approximately twice as large in state II. Measurements at low temperature also showed that the cross sections and the spectral properties of the photosystems did not change in the transitions between state I and state II. In agreement with predictions made from the parameters measured at low temperature, the action spectra for oxygen evolution measured at room temperature were found to be the same in state I and state II.

Diuron↗
PubMed1980

A study of dark luminescence in Chlorella. Background luminescence, 3-(3,4-dichlorophenyl)-1,1-dimethylurea-triggered luminescence and hydrogen peroxide chemiluminescence.

Dark luminescence, defined as the ability of completely relaxed (dark-adapted) photosynthetic systems to emit light, has been studied in Chlorella. Three main effects have been demonstrated. 3-(-3,4-Dichlorophenyl)-1,1-dimethylurea elicits a weak emission LD of very long lifetime (several minutes); it is believed to result from a negative shift of redox potential of the secondary System II electron acceptor B producing in some centers a state Q- (reduced primary acceptor), as postulated by Velthuys and Amesz ((1974 Biochim. Biophys. Acta 333, 85--94), which can recombine with an oxidizing equivalent in a state S2 present in very small amount. As in photoinduced luminescence, this recombination excites chlorophyll which then emits light. A much stronger emission LH is observed after injection of H2O2. Both signals are modified or suppressed by treatments specific of the oxygen emission system, such as: thermal denaturation at 50 degrees C, NH2OH, etc. In addition, a weak, permanent background luminescence L0 has been observed; like LD and and LH, it is a System II property and requires the integrity of the oxygen-evolving system. It is believed to reflect a very slow back flow of electrons from an endogeneous reductant pool to oxygen through part of the photosynthetic chain. Using flash preillumination, it is demonstrated that H2O2 is able to oxidize S0 into S2, the latter giving rise to LH; H2O2 does not act on S1 (or much less). The reactive site of H2O2 seems to be the same as the binding site of NH2OH. Evidence is given that the strong LH signal in particular reveals a stable, low pH of the intrathylakoid phase in Chlorella.

Chlorella↗
PubMed1980

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites in photosystem II of spinach chloroplasts.

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites were found in PS II of spinach chloroplasts, depending on the pH of the assay medium used. The low site (pH 6) can be inhibited by certain quinolines, such as 8-hydroxyquinoline at concentrations less than 50 microM. The high pH site (pH 8) can be inhibited by disodium cyanamide, folic acid, or 5,6-benzoquinoline at concentrations from 50 microM to 5 mM. With the exception of orthophenanthroline, which stimulates the high pH site but does not show much inhibition at low pH, all other inhibitors gave opposite effects at the pH values used, i.e., they stimulated at low pH or inhibited at high pH, or vice versa. Several mechanisms for the observed effects are discussed.

Ammonium Chloride↗
PubMed1981

Investigation of double turnovers in photosystem II charge separation and oxygen evolution with excitation flashes of different duration.

The characteristics of double hitting in Photosystem II charge separation and oxygen evolution in algae and chloroplasts were investigated with saturating excitation flashes of 3 microseconds, 300 ns and 5 ns duration. Two types of double hitting or advancement in S-states were found to occur in oxygen evolution: a non-photochemical type found even with 5 ns flashes and a photochemical type seen only with microsecond-long flashes, which have extensive tails. The non-photochemical type, occurring with a probability of about 3%, is sensitive to the physiological condition of the sample, and is only present in algae or chloroplast samples that have been freshly prepared. In chloroplasts incubated with ferricyanide, a 3-fold increase in double advancement of S-states is observed with xenon-flash illumination but not with 300 ns or 5 ns laser illumination. However, double turnovers in Photosystem II reaction center charge separation are large with xenon flash or 300 ns laser illumination but not with 5 ns laser illumination. This indicates that quite different kinetic processes are involved in double advancement in S-states for oxygen evolution and double turnovers in charge separation. Various models of the Photosystem II reaction center are discussed. Also, based on experiments with chloroplasts incubated with ferricyanide, an unique solution to the oxygen S-state distribution in the dark suggested by Thibault (Thibault, P. (1978) C.R. Acad. Sci. Paris 287, 725-728) can be rejected.

Chlorella↗
PubMed1981

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5'-triphosphate.

Addition of ATP to chloroplasts causes a reversible 25-30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at -196 degrees C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F0) and variable (Fv) fluorescence such that the ratio of Fv to the maximum (Fm) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of Fv against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.

Adenosine Triphosphate↗
PubMed1981

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation.

The kinetics of Mn release during NH2OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH2OH. This appears to reflect reduction by NH2OH of higher S-states to a common more reduced state (S0 or S-1) which alone is susceptible to NH2OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2--5 S-1 effectively prevent NH2OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 S. Under continuous illumination, protection against NH2OH inactivation is maximally effective at intensities in the range 10(3)--10(4) erg . cm-2 . S-1. This behavior differs from that of NH2OH-induced Mn release, which is strongly inhibited at all intensities greater than 10(3) erg . cm-2 . S-1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH2OH liberates manganese, although the overall process of water oxidation is inactivated by NH2OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 microM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1981

External electric field effects on prompt and delayed fluorescence in chloroplasts.

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233-240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulate luminescence supported the interpretation given above. Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.

Chloroplasts↗