Different kinetics of membrane potential formation in dark-adapted and preilluminated chloroplasts

The effects of varying dark interval on the kinetics of light-induced formation of the membrane potential were studied on individual chloroplasts of Anthoceros with the use of capillary microelectrodes. Illumination of the chloroplast with 1 s light pulse after 3 min dark period induced the photoelectrical response with two peaks of the potential that were located at 20 and 500 ms after the onset of illumination. The position of the second peak was shifted along the time-scale depending on the preceding dark interval. The repeated illumination of the chloroplast with 1 s light pulse after 30 s dark interval induced the electrical response with only one maximum and a monotonous decay of the potential in the light. Distinctions in the electrical responses induced by the first and the second light pulses were eliminated by the addition of 50 μM dicyclohexylcarbodiimide (DCCD). The results show that the photoinduction kinetics of the membrane potential in chloroplasts is affected by functioning of H⁺-ATPase. The delayed peak of the membrane potential in the photoinduction kinetics is interpreted as a consequence of the photoactivated electron transport supported by Photosystem I.     

‘Low-waves>’ in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate*

A brief reversible lowering of chlorophyll fluorescence yield (so called low-waves) immediately after application of a saturating light pulse in parallel with a short-time enhancement of the P700 oxidation level was observed in the green alga Haematococcus pluvialis. The phenomenon occurred in the steady-state time region of fluorescence induction kinetics under mild acidic conditions, and was eliminated by bicarbonate. Shortly after expression of low-waves, the photosynthetic oxygen evolution rate decreased and the non-photochemical chlorophyll fluorescence quenching component increased. The enhancement of the non-photochemical chlorophyll fluorescence quenching component was nigericin-sensitive indicating its dependence on the transthylakoid proton gradient. On the other hand, the formation of low-waves was not removed by the uncoupler. Only when bicarbonate was applied additionally, the reversible short-term decrease in fluorescence yield following each saturating light flash was abolished. Dimethyl-4-nitroso-aniline as an artificial electron acceptor of Photosystem I did not limit the brief drops in fluorescence. However, formate as a competitive inhibitor of bicarbonate binding in Photosystem II induced low-wave formation. Therefore, our results suggest that low-waves in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate in the reaction centre of Photosystem II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acid-base status in plasma of trout and eel in hypocapnic and normocapnic conditions

Summary Under certain conditions, circadian locomotor activity of the cockroachesLeucophaea maderae (Fabr.) andBlaberus fuscus (Burm.) exhibits two peaks per cycle. Phase-shifting experiments with split rhythms inL. maderae show an identical reaction in both peaks, revealing a strong coupling. Light pulses evoke either delays or advances dependent on the phase of exposure. Phase shifts of bimodal rhythms fit the shape of the phase response curve derived from unimodal systems. Thus the first peak is decisive while the second one shifts accordingly, remaining in a constant phase angle with the other. The results suggest that the bimodal activity pattern inL. maderae isnot a result of two pacemakers each driving one activity peak, as is generally proposed for splitting. Instead, a unitary oscillator system is postulated which shows identical phase shifting properties when driving either a unimodal activity pattern or two peaks of a bimodal rhythm.Abbreviations LD light dark cycle - LL continuous white light - LP light pulse - LTP pulse of low temperature - PRC phase response curve - RR continuous red light - fre-running period I am very grateful to Dr. Wolfgang Engelmann for providing the facilities in the laboratory, which made this publication possible. I thank him, Larry Orsak, and Drs. M.K. Chandrashekaran, Anders Johnsson, Werner Loher, and Hugh Rowell for helpful discussion and critical reading of the paper. This study has been supported by grants of the Deutsche Forschungsgemeinschaft to Dr. Wolfgang Engelmann.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.     

An Analysis of the Mechanism of the Low-wave Phenomenon of Chlorophyll Fluorescence

The low-wave phenomenon, i.e., the transient drop of yield of modulated chlorophyll fluorescence shortly after application of a pulse of saturating light, was investigated in intact leaves of tobacco and Camellia by measuring fluorescence, CO(2) assimilation and absorption at 830 nm simultaneously. Limitations on linear electron flow, due to low electron acceptor levels that were induced by low CO(2), induced the low waves of chlorophyll fluorescence. Low-wave amplitudes obtained under different CO(2) concentrations and photon-flux densities yielded single-peak curves when plotted as functions of fluorescence parameters such as PhiPS II (quantum yield of Photosystem II) and qN (coefficient of non-photochemical quenching), suggesting that low-wave formation depends on the redox state of the electron transport chain. Low waves paralleled redox changes of P700, the reaction center of Photosystem I (PS I), and an additional electron flow through PS I was detected during the application of saturating pulses that induced low-waves. It is suggested that low waves of chlorophyll fluorescence are induced by increased non-photochemical quenching, as a result of the formation of a trans-thylakoid proton gradient due to cyclic electron flow around PS I.     

Toxic and non-toxic Nodularia strains can be distinguished from each other and from eukaryotic algae with chlorophyll fluorescence fingerprinting

Chlorophyll fluorescence induction curves of toxic and non-toxic strains of the cyanobacterium Nodularia were measured and compared with fluorescence curves measured from four species of eukaryotic algae. Both cyanobacteria and algae were isolated from the Baltic Sea. The results show that Nodularia strains can be distinguished from the eukaryotes by applying a pattern recognition procedure to the fluorescence induction curves, suggesting that the fluorescence fingerprinting technique might be useful in environmental monitoring of marine algae. The six studied Nodularia strains could not be distinguished from each other from their fluorescence induction kinetics. However, their fluorescence curves fell into two clear categories, the toxic and the non-toxic Nodularia. Emission spectroscopy and differences in the fluorescence induction curves showed that the ratio of the intensity of the Photosystem I emission peak to the Photosystem II peak is higher in non-toxic Nodularia than in the toxic strains, suggesting that the toxicity affects the structure of the photosynthesis machinery. The effect on photosynthesis may be related to the ability of the microcystins to chelate iron.     

Temporal analysis of tone pulses within the courtship songs of two siblingDrosophila species,their interspecific hybrid,and behavioral mutants ofD. melanogaster (Diptera: Drosophilidae)

Temporal analyses were applied to the tone pulses within the courtship songs of Drosophila melanogaster, D. simulans,their interspecific hybrid, and behavioral mutants of D. melanogaster.Linear regression was performed on various parameters of the song pulses (cycles per pulse, absolute peak amplitude, intrapulse frequency, number of peaks in fast Fourier transform, width of the primary frequency peak, and interpulse interval), as a function of their positions within pulse trains. Significant differences in the slope values of these two species and of the mutant genotypes allowed for discriminative quantification of temporal changes within trains. These results are discussed in relation to previous kinds of temporal analyses of Drosophilacourtship songs and also with regard to the mechanisms of song production.     

Photoresponses of Halobacterium salinarum to repetitive pulse stimuli.

Halobacterium salinarum cells from 3-day-old cultures have been stimulated with different patterns of repetitive pulse stimuli. A short train of 0.6-s orange light pulses with a 4-s period resulted in reversal peaks of increasing intensity. The reverse occurred when blue light pulses were delivered as a finite train: with a 3-s period, the response declined in sequence from the first to the last pulse. To evaluate the response of the system under steady-state conditions of stimulation, continuous trains of pulses were also applied; whereas blue light always produced a sharply peaked response immediately after each pulse, orange pulses resulted in a declining peak of reversals that lasted until the subsequent pulse. An attempt to account for these results in terms of current excitation/adaptation models shows that additional mechanisms appear to be at work in this transduction chain.     

Photosystem I-dependent cyclic electron transport is important in controlling Photosystem II activity in leaves under conditions of water stress

Leaves of the C₃ plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO₂ and/or oxygen. In the absence of CO₂, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P₅₁₅ signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO₂ inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background to red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO₂ levels were decreased to or below the CO₂ compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F₀ fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor Q_A in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.Abbreviations F, F_M, F'_M, F"_M, F₀, F'₀ chlorophyll fluorescence levels - _exc quantum efficiency of excitation energy capture by open Photosystem II - _{PS II} quantum efficiency of electron flow through Photosystem II - P₅₁₅ field indicating rapid absorbance change peaking at 522 nm - P₇₀₀ primary donor of Photosystem I - Q_A primary quinone acceptor in Photosystem II - Q_N non-photochemical fluorescence quenching - Q_q photochemical quenching of chlorophyll fluorescence     

Physiological and biochemical plasticity of Lepidium latifolium as ‘sleeper weed’ in Western Himalayas

To understand the spread of native populations of Lepidium latifolium growing in different altitudes in Ladakh region of Western Himalayas, photosynthetic and fluorescence characteristics were evaluated in relation to their micro‐environment. Three sites representing sparsely populated (SPS), moderately populated (MPS) and densely populated site (DPS) were selected. Results showed that the DPS had higher photosynthetic accumulation than MPS and SPS. The higher transpiration rate at DPS despite lower vapor pressure deficit and higher relative humidity suggest the regulation of its leaf temperature by evaporative cooling. Intrinsic soil parameters such as water holding capacity and nutrient availability also play crucial role in higher biomass here. The quantum efficiency of PSII photochemistry (F_v/F_m, non‐photochemical quenching (NPQ), Φ_PSII) and light curve at various PPFDs suggests better light harvesting potential and light compensation point at DPS than the other two sites. Concomitantly, plants at SPS had significantly higher lipid peroxidation, suggesting a stressful environment, and higher induction of antioxidative enzymes. Metabolic content of reduced glutathione also suggests an efficient mechanism in DPS and MPS than SPS. High light intensities at MPS are managed by specialized contrive of carotenoid pigments and PsbS gene product. Large pool of violaxanthin and lutein plays an important role in this response. It is suggested that L. latifolium is present as ‘sleeper weed’ that has inherent biochemical plasticity involving multiple processes in Western Himalayas. Its potential spread is linked to site‐specific micro‐environment, whereby, it prefers flat valley bottoms with alluvial fills having high water availability, and has little or no altitudinal effect.     

Concentrations of circulating hormones during the interval between pulses of a PGF2α metabolite in mares and heifers

The temporal relationship of several hormones to a metabolite of prostaglandin F2α (PGFM) was studied in mares and heifers from the beginning of the first PGFM pulse during luteolysis to the end of the second pulse. Mares (n=7) were selected with a 9-h interval between the peaks of the two pulses. In mares, estradiol-17β (estradiol) increased (P<0.05) within each PGFM pulse and plateaued for a mean of 6h between the pulses, resulting in a stepwise estradiol increase. Progesterone decreased linearly (P<0.0001) throughout the intra-pulse and inter-pulse intervals of PGFM. In heifers (n=6), inter-pulse intervals were variable, and therefore Hours 1-4 of the first pulse (Hour 0=PGFM peak) and Hours -4 to -1 of the second pulse were used to represent the mean 8-h interval between peaks of the two pulses. Estradiol increased (P<0.05) during the ascending portion of each PGFM pulse and then decreased (P<0.05) beginning at Hour -1 of the first PGFM pulse and Hour 0 of the second pulse. The 1-h delay during the second pulse was accompanied by an apparent increase in PRL. A transient decrease in estradiol occurred in individuals between PGFM pulses at a mean of 5h after the first PGFM peak, concomitant with a transient LH increase (P<0.05). Results indicated that estradiol plateaued in mares and fluctuated in heifers during the interval between PGFM pulses. Heifers also showed temporal relationships between estradiol and LH and apparently between estradiol and PRL.     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectiely) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 μmol O₂/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (?196°C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O₂/einstein (605 nm), with a lesser change in the V_max values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Studies on thermoluminescence,delayed light emission and oxygen evolution from photosynthetic materials: UV effects

The effect of ultraviolet light on thermoluminescence, oxygen evolution and the slow component of delayed light has been investigated in chloroplasts and Pothos leaves. All peaks including peak V (48°C) were inhibited by UV. However, the peak at 48°C which was induced by DCMU was enhanced following UV irradiation of chloroplasts at ambient temperature (23°C) whereas peak II (-12°C) and peak III (10°C) which were also induced by DCMU were inhibited. Chloroplasts treated with DCMU and dark incubated for several minutes at ambient temperature prior to recording of glow curves have also shown enhancement of peak at 48°C. A slow component of delayed light and photosystem II activity of chloroplasts were inhibited by UV whereas photosystem I activity was marginally affected. These results corroborate involvement of photosystem II in generating thermoluminescence and slow components of delayed light in photosynthetic materials.Abbreviations DCIP Dichlorophenol Indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCQ 2,6 Dichloro-p-benzoquinone - DLE delayed light emission - MOPS Morpholino propane sulfonic acid - PSI Photosystem I - PS II Photosystem II - TL thermoluminescence     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis.

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectively) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 mumol O2/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (-196 degrees C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O2/einstein (605 nm), with a lesser change in the Vmax values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Cell membrane electroporator with digital generation of random shaped pulses

A Digital Poration System (DPS), a versatile device for electrotreatment of biological objects by electric field pulses; was designed, constructed, and implemented. A feature distinguishing DPS from the currently available electroporators based on capacitor discharge through the load is the use of a digital-to-analog converter card as a generator of pulses applied for electroporation of biological membranes, with further amplification of the pulse by both voltage and current. The shape of pulses, including bipolar pulses, is arbitrarily programmable in DPS unlike other electroporators providing exponentially decaying and square-wave pulses only. Thus, the application area of DPS is substantially extended. In DPS, many of the drawbacks inherent in capacitor electroporators are removed, including the need for an additional external pulse analyzer monitoring and logging the electroporation processes, the necessity to recharge the capacitor before any new pulse, a poor precision of setting and measuring the pulse parameters, the need for an additional generator of long-lasting low-voltage signals for electrophoresis of ions into the porated object, the need for additional AC generators for the alignment of cells before, after, and during electroporation, and the need for an additional microprocessor to control multi-pulse and/or repetitive protocols. DPS provides a slew rate of about 1 V/1 ns required for the electroporation of most mammalian somatic cells, with +/- 250 V output voltage and 500 Ohm load resistance. The application area of DPS is much wider than for the available porators. It includes electrochemotherapy, cell electrofusion, oocyte activation by mimicking calcium waves (the latter two are the crucial components of mammalian organism cloning technology), dielectrophoretic bunching and orientation ordering of cells, sorting of cells, and electrophoresis of charged species into the cells.     

KINETICS OF BLUE-LIGHT STIMULATION AND CIRCADIAN RHYTHMICITY OF LIGHT-SATURATED PHOTOSYNTHESIS IN BROWN ALGAE: A SPECIES COMPARISON1

The time courses of photosynthetic rates in red light, with and without additional blue light, were investigated and compared in 20 species of brown algae. Species could be separated into two groups on the basis of the rhythmicity of their photosynthesis in red light and the kinetics of their responses to blue-light pulses. One group, which consisted of members of the Ectocarpales, Chordariales, and Dictyosiphonales, was characterized by strong and persistent circadian rhythmicity in red light. The photosynthetic responses of these species to blue-light pulses started within 10–30 s of the beginning of blue-light treatment and mostly contained at least two distinct kinetic components. An early component, which reached a maximum about 5–10 min after the blue-light pulse, was always detectable. Later components were seen as separate peaks or shoulders after an additional 10–20 min. The decay of the response in this group of species was mostly slow, with half-lives of between 0.5 and 1.5 h. In the second group of species, consisting of members of the Dictyotales, Laminariales, and Fucales, photosynthesis in red light was usually non-rhythmic, although circadian rhythms with a weak amplitude or of transient occurrence were observed in some plants of some species. The increase in photosynthesis in response to a blue-light pulse was not detectable until 70–330 s after the start of blue-light treatment, and the response itself had only a single component, with a maximum after about 10 min and half-life of 10–20 min. The lengths of the lag-phases were positively correlated with the times taken to reach the peak in this group, although the lag-phases and the half lives sometimes varied with time in individual plants. Two members of the Sphacelariales (Sphacelaria, Cladostephus) did not fit into either of the two groups because their photosynthesis was rhythmic, but their responses had long lag-phases, a single component, and moderately long half-lives. The differences in the kinetics of the photosynthetic response to blue-light pulses, which have been described for the two main groups of species, are thought to indicate that there are two distinct mechanisms by which light-saturated photosynthesis responds to blue light in brown algae. Since in some species the maximal photosynthesis after a blue-light pulse and the rate of photosynthesis in continuous blue light also varied in a circadian pattern, the response to blue light itself may be under circadian control.     

Parallel inductive kinetics of fluorescence and photoacoustic signal in dark-adapted thalli of Bryopsis maxima

The inductive kinetics of fluorescence and photoacoustic signal were measured simultaneously in dark-adapted thalli of the green coenocytic alga Bryopsis maxima. Under illumination with weak red light modulated at 60 Hz, the fluorescence yield varied, showing three maxima P, M₁ and M₂ almost immediately, 10 s and 6 min after the onset of the illumination, respectively (Yamagishi, A., Satoh, K. and Katoh, S. (1978) Plant Cell Physiol. 19, 17–25). The photoacoustic signal also showed inductive transients which parallel well those of the fluorescence up to the M₂ stage. After M₂, the photoacoustic signal remained at a constant level, while the emission yield gradually decreased. The first peak of the fluorescence induction and a corresponding peak of the photoacoustic transients were selectively eliminated by prior illumination or methyl viologen treatment of the dark-adapted thalli. The second peaks of the two induction curves were abolished by carbonylcyanide-m-chlorophenylhydrazone, whereas dicyclohexylcarbodiimide enhanced their peak heights and suppressed the subsequent decreases. The results indicate that the fluorescence yield is mainly determined by the redox state of the Photosystem II reaction center throughout the induction period except the last phase. Mechanisms underlying inductive transients of fluorescence are discussed in the light of the present findings.     

Photosynthesis in Chondrus crispus: The contribution of energy spill-over in the regulation of excitonic flux

Chondrus crispus is a species of red algae that grows on rocks from the middle intertidal into the subtidal zones of the North Atlantic coasts. As such, it has to cope with strongly variable abiotic conditions. Here we studied the response of the photosynthetic apparatus of this red alga to illumination. We found that, as previously described in the case of the unicellular alga Rhodella violacea (E. Delphin et al., Plant Physiol. 118 (1998) 103–113), a single multi-turnover saturating pulse of light is sufficient to induce a strong quenching of fluorescence. To elucidate the mechanisms underlying this fluorescence quenching, we combined room temperature and 77 K fluorescence measurements with absorption spectroscopy to monitor the redox state of the different electron carriers in the chain. In addition, we studied the dependence of these various observables upon the excitation wavelength. This led us to identify energy spill-over from Photosystem II to Photosystem I rather than a qE-type non-photochemical quenching as the major source of fluorescence quenching that develops upon a series of 200 ms pulses of saturating light results, in line with the conclusion of Ley and Butler (Biochim. Biophys. Acta 592 (1980) 349–363) from their studies of the unicellular red alga Porphyridium cruentum. In addition, we show that the onset of this spill-over is triggered by the reduction of the plastoquinone pool.     

Probing Photosynthesis on a Picosecond Time Scale: Evidence for Photosystem I and Photosystem II Fluorescence in Chloroplasts

Fluorescent emission kinetics of isolated spinach chloroplasts have been observed at room temperature with an instrument resolution time of 10 ps using a frequency doubled, mode-locked Nd:glass laser and an optical Kerr gate. At 685 nm two maxima are apparent in the time dependency of the fluorescence; the first occurs at 15 ps and the second at 90 ps after the flash. The intervening minimum occurs at about 50 ps. On the basis of theoretical models, lifetimes of the components associated with the two peaks and spectra (in escarole chloroplasts), the fluorescence associated with the first peak is interpreted as originating from Photosystem I (PSI) (risetime ≤10 ps, lifetime ≤10 ps) and the second peak from Photosystem II (PSII) (lifetime, 210 ps in spinach chloroplasts and 320 ps in escarole chloroplasts). The fact that there are two fluorescing components with a quantum yield ratio ≤0.048 explains the previous discrepancy between the quantum yield of fluorescence measured in chloroplasts directly and that calculated from the lifetime of PSII. The 90 ps delay in the peak of PSII fluorescence is probably explained by energy transfer between accessory pigments such as carotenoids and Chl a. Energy spillover between PSI and PSII is not apparent during the time of observation. The results of this work support the view that the transfer of excitation energy to the trap complex in both photosystems occurs by means of a molecular excitation mechanism of intermediate coupling strength. Although triplet states are not of major importance in energy transfer to PSII traps, the possibility that they are involved in PSI photochemistry has not been eliminated.