Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl2-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3',4'-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a pico-second time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp --At1/2, where A was found to be 0.052 ps-1/2, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl2 produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence deday law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

The lipid composition of a barley mutant lacking chlorophyll b.

The acyl-lipid composition of a barley mutant that contained no detectable chlorophyll b was studied. This mutant contained chloroplasts that were much less organized than chloroplasts of normal barley. The mutant contained all the normal acyl lipids, with small increases in the relative concentration of phosphatidylglycerol and diacylsulphoquinoglycerides was unchanged, but most other lipids of the mutant barley contained lower amounts of alpha-linolenic acid compared with normal. There was no difference in the transhexadec-3-enoic acid content of phosphatidylglycerol, which was evidence against this lipid being involved in grana stacking.     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.     

Picosecond time-resolved fluorescence studies on excitation energy transfer in a histidine 117 mutant of the D2 protein of photosystem II in Synechocystis 6803

The role of the peripheral reaction center chlorophyll a molecule associated with His117 of the D2 polypeptide in photosystem II was investigated in Synechocystis sp. PCC 6803 using a combination of steady state, pump-probe, and picosecond time-resolved fluorescence spectroscopy. Data were obtained from intact cells and isolated thylakoid membranes of a control mutant and a D2-H117T mutant, both of which lacked photosystem I. Excitation energy transfer and trapping were investigated by analyzing the data with a kinetic model that used an exact numerical solution of the Pauli master equation, taking into account available photosystem II spectral and structural information. The results of our kinetic analysis revealed the observed difference in excited-state dynamics between the H117T mutant and the control to be consistent with a retardation of the rate of excitation energy transfer from the peripheral chlorophyll of D2 (Chl at His117) to the electron-transfer pigments and an increase of the rate constant for charge recombination in the H117T mutant. The kinetic model was able to account for the experimentally observed changes in absorption cross section and fluorescence decay kinetics between the control and mutant by invoking changes in only these two rate constants. The results rule out quenching of excitation by a chlorophyll cation radical as a mechanism responsible for the lower efficiency of excitation energy utilization in the H117T mutant. Our work also demonstrates the importance of the chlorophyll associated with His117 of the D2 protein for excitation energy transfer to the PSII electron-transfer pigments and for the effective stabilization of the primary radical pair.     

Cation-mediated regulation of excitation energy distribution in chloroplasts lacking organized Photosystem II complexes

Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll $\text{a}\text{b}$ pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg²⁺-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg²⁺ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg²⁺ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg²⁺ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg²⁺ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg²⁺-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.     

Chlorophyll fluorescence transients in a barley mutant lacking Photosystem I

We have compared the properties of a mutant of barley lacking Photosystem I (viridis-zb ⁶³ ) with the corresponding wild type using modulated fluorescence measurements. The mutant showed two unexpected characteristics. Firstly, there was a slow decline in the fluorescence signal in the light which was dependent on the presence of O₂ at concentrations similar to that in air; 2% O₂ in N₂ had no effect. The observed decline was mainly due to an increase in the non-photochemical quenching. Secondly, in the absence of O₂, saturating light pulses caused a pronounced transient decrease in the fluorescence signal; a similar effect could also be observed in wild type plants when neither CO₂ nor O₂ was present.Abbreviations PPFD- photosynthetic photon flux density - q_N- non-photochemical quenching of chlorophyll fluorescence - q_p- photochemical quenching of chlorophyll fluorescence     

Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae

Single-photon timing with picosecond resolution is used to investigate the kinetics of the fluorescence emission of chlorophyll a in chloroplasts from spinach and pea and in the algae Chlorella pyrenoidosa and Chlamydomonas reinhardii. The fluorescence decay is best described by three exponential components in all species. At low light intensity and with open reaction centers of Photosystem II (F₀), we find lifetimes of approx. 100, 400 and 1100 ps for the three components. Closing the reaction centers by addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea plus hydroxylamine and by increasing light intensity produces only minor changes in the almost constant fast- and medium-lifetime components; however, there is a dramatic increase in the yield of the slow component, by a factor of about 20, accompanied by only a modest increase in the lifetime to 2200 ps (F_max). In good agreement with previous fluorescence lifetime measurements, we find an increase in the averaged lifetime of the three components from 0.5 to 2.0 ns, which is proportional to the 4-fold increase in the total fluorescence yield. Our time-resolved results are inconsistent with models which are based on the proportionality between lifetime and yield and which involve a homogeneous origin of fluorescence that is sensitive to the state of the reaction centers. We conclude that the variable part of the fluorescence, which is dominated by the slow phase, reflects the kinetics of charge recombination in the reaction center, as proposed previously (Klimov, V.V., Allakhverdiev, S.I. and Paschenko, V.Z. (1978) Dokl. Akad. Nauk S.S.S.R. 242, 1204–1207). The modest increase in lifetime of the slow phase indicates the presence of some energy transfer between photosynthetic units.     

Immunological evidence for apoproteins of the light-harvesting chlorophyll-protein complex in a mutant of barley lacking chlorophyll b

A barley mutant lacking chlorophyll b and the pigmented light-harvesting chlorophyll-protein of photo-system 2 is shown by several criteria to contain functional apoproteins of the light-harvesting complex. 1. Electrophoretic comparison of thylakoid polypeptide patterns, and the effects of trypsin treatment on these, suggests that the mutant contains several polypeptides equivalent in mobility to those of the wild-type complex. 2. An antibody monospecific for the light-harvesting complex agglutinated both wild-type and mutant thylakoids. 3. 'Western blot' immunoelectrophoretic analysis indicated that of four distinct subunits of the light-harvesting complex in the wild-type thylakoids, three are detectable in the mutant. 4. As in wild-type lamellae at least one of the light-harvesting complex polypeptides is phosphorylated by the endogenous protein kinase. The results are considered in terms of a general role for the light-harvesting complex polypeptides in membrane appression and the regulation of excitation energy distribution within thylakoids.     

Low-Intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts

The fluorescence decays of barley chloroplasts have been measured by single-photon counting with tunable picosecond dye laser excitation. The fluorescence decays of dark-adapted chloroplasts are best fitted to a sum of three exponential lifetime components with lifetimes of 112, 380 and 2214 ps. The relative magnitude of each component is shown to be dependent on the excitation wavelength and collected emission wavelength. The excitation wavelength dependence is correlated with the Photosystem (PS) I and PS II action study of Ried [36] and with the measured pigment distributions in the photosynthetic unit [37,41]. Experiments varying the single excitation pulse intensity from 10⁸ to 10¹² photons/cm² pulse show that our results are not distorted by singlet-singlet annihilation. Unflowed samples where the cloroplasts are under constant illumination show 2-fold increases in quantum yield of fluorescence primarily in the two longer lifetime components. Theoretical calculations of Shipman [31] on an isolated reaction center with a homogeneous antenna are discussed and the principles extended to discussion of the measured barley chloroplast fluorescence decay components in terms of photosynthetic unit light-harvesting array models and earlier experimental work. Our data support a photosynthetic unit model in which 70–90% of the photons absorbed are quenched by either PS I or efficiently quenching PS II in a process where the fluorescence lifetime is 100 ps. The origin of the intermediate 380 ps. component is probably due to excitation transfer to a PS II reaction center in a redox state which quenches less efficiently.     

Excitation energy transfer in thylakoid membranes from Chlamydomonas reinhardtii lacking chlorophyll b and with mutant Photosystem I

Energy trapping in Photosystem I (PS I) was studied by time-resolved fluorescence spectroscopy of PS II-deleted Chl b-minus thylakoid membranes isolated from site-directed mutants of Chlamydomonas reinhardtii with specific amino acid substitutions of a histidine ligand to P₇₀₀. In vivo the fluorescence of the PS I core antenna in mutant thylakoids with His-656 of PsaB replaced by asparagine, serine or phenylalanine is characterized by an increase in the lifetime of the fast decay component ascribed to the energy trapping in PS I (25 ps in wild type PS I with intact histidine-656, 50 ps in the mutant PS I with asparagine-656 and 70 ps in the mutant PS I with phenylalanine-656). Assuming that the excitation dynamics in the PS I antenna are trap-limited, the increase in the trapping time suggests a decrease in the primary charge separation rate. Western blot analysis showed that the mutants accumulate significantly less PS I than wild type. Spectroscopically, the mutations lead to a decrease in relative quantum yield of the trapping in the PS I core and increase in relative quantum yield of the fluorescence decay phase ascribed to uncoupled chlorophyll–protein complexes which suggests that improper assembly of PS I and LHC in the mutant thylakoids may result in energy uncoupling in PS I.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

Studies on chlorophyll fluorescence in chloroplasts. I. Effects of dithionite on fluorescence of chlorophyll A in isolated spinach chloroplasts

Effects of dithionite on the time-course of fluorescence emitted from chlorophyll a in isolated spinach chloroplasts were studied. Addition of dithionite markedly shortened the induction period of fluorescence and increased the steady-state level of fluorescence. However, a small but distinct induction, comparable to that observed in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea, was always observed in the presence of dithionite. When the fluorescence change was determined in the presence of DCMU, preincubation of the chloroplasts with dithionite for a prolonged period further shortened, but only slowly, the induction period. However, addition of DCMU during the incubation period abolished most of the effects of dithionite in reducing the induction period. The results obtained were interpreted in terms of the reduction by dithionite of endogenous electron carriers associated with photosystem 2.