Exploring the mechanism(s) of energy dissipation in the light harvesting complex of the photosynthetic algae Cyclotella meneghiniana

Photosynthetic organisms have developed vital strategies which allow them to switch from a light-harvesting to an energy dissipative state at the level of the antenna system in order to survive the detrimental effects of excess light illumination. These mechanisms are particularly relevant in diatoms, which grow in highly fluctuating light environments and thus require fast and strong response to changing light conditions. We performed transient absorption spectroscopy on FCPa, the main light-harvesting antenna from the diatom Cyclotella meneghiniana, in the unquenched and quenched state. Our results show that in quenched FCPa two quenching channels are active and are characterized by differing rate constants and distinct spectroscopic signatures. One channel is associated with a faster quenching rate (16 ns^− 1) and virtually no difference in spectral shape compared to the bulk unquenched chlorophylls, while a second channel is associated with a slower quenching rate (2.7 ns^− 1) and exhibits an increased population of red-emitting states. We discuss the origin of the two processes in the context of the models proposed for the regulation of photosynthetic light-harvesting. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana

Thylakoids of the diatom Cyclotella meneghiniana were separated by discontinuous gradient centrifugation into photosystem (PS) I, PSII, and fucoxanthin-chlorophyll protein (FCP) fractions. FCPs are homologue to light harvesting complexes of higher plants with similar function in e.g. brown algae and diatoms. Still, it is unclear if FCP complexes are specifically associated with either PSI or PSII, or if FCP complexes function as one antenna for both photosystems. However, a trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have been described for C. meneghiniana, already. In this study, biochemical and spectroscopical evidences are provided that reveal a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PSII associated Fcp antenna resembles FCPa since it contains Fcp2 and Fcp6, at least three different Fcp polypeptides are associated with PSI. By re-solubilisation and a further purification step Fcp polypeptides were partially removed from PSI and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby a protein related to Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PSI, whereas presumably Fcp5, a subunit of the FCPb complex, is only loosely bound to the PSI core. Thus, an association of FCPb and PSI is assumed.     

Stark fluorescence spectroscopy reveals two emitting sites in the dissipative state of FCP antennas

Diatoms are characterized by very efficient photoprotective mechanisms where the excess energy is dissipated as heat in the main antenna system constituted by fucoxanthin–chlorophyll (Chl) protein complexes (FCPs). We performed Stark fluorescence spectroscopy on FCPs in their light-harvesting and energy dissipating states. Our results show that two distinct emitting bands are created upon induction of energy dissipation in FCPa and possibly in FCPb. More specifically one band is characterized by broad red shifted emission above 700 nm and bears strong similarity with a red shifted band that we detected in the dissipative state of the major light-harvesting complex II (LHCII) of plants [26]. We discuss the results in the light of different mechanisms proposed to be responsible for photosynthetic photoprotection.     

Properties of zeaxanthin and its radical cation bound to the minor light-harvesting complexes CP24, CP26 and CP29

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis by which plants protect themselves against excess excitation energy and which is thus of crucial importance for plant survival and fitness. Recently, carotenoid radical cation (Car^•+) formation has been discovered to be a key step in the feedback deexcitation quenching component (qE) of NPQ, whose molecular mechanism and location remains elusive. A recent model for qE suggests that the replacement of violaxanthin (Vio) by zeaxanthin (Zea) in photosynthetic pigment binding pockets can in principle result in qE via the so-called “gear-shift” or electron transfer quenching mechanisms. We performed pump-probe measurements on individual antenna complexes of photosystem II (CP24, CP26 and CP29) upon excitation of the chlorophylls (Chl) into their first excited Q_y state at 660 nm when either Vio or Zea was bound to those complexes. The Chl lifetime was then probed by measuring the decay kinetics of the Chl excited state absorption (ESA) at 900 nm. The charge-transfer quenching mechanism, which is characterized by a spectral signature of the transiently formed Zea radical cation (Zea^•+) in the near-IR, has also been addressed, both in solution and in light-harvesting complexes of photosystem II (LHC-II). Applying resonant two-photon two-color ionization (R2P2CI) spectroscopy we show here the formation of β-Car^•+ in solution, which occurs on a femtosecond time-scale by direct electron transfer to the solvent. The β-Car^•+ maxima strongly depend on the solvent polarity. Moreover, our two-color two-photon spectroscopy on CP29 reveals the spectral position of Zea^•+ in the near-IR region at 980 nm.     

On photoprotective mechanisms of carotenoids in light harvesting complex

Carotenoids in light harvesting complex (LHC) play an important role in preventing plants photodamage caused by excess light. Non-photochemical quenching (NPQ) is an important mechanism adopted by plants to deal with high light intensity and the major component is referred to as energy dependent quenching (qE). Despite numerous studies have been devoted to investigating the site and mechanism of qE, there are still much debate on these topics. In this article, we discussed the possible site and underlying mechanism of qE based on the structural similarity of carotenoids. Moreover, being as good antioxidants, carotenoids’ potential protective effects against LHC photo-oxidation by quenching active oxygen species or triplet excited state chlorophyll are also discussed.     

Oligomerization and pigmentation dependent excitation energy transfer in fucoxanthin-chlorophyll proteins

The ultrafast caroteonid to chlorophyll a energy transfer dynamics of the isolated fucoxanthin-chlorophyll proteins FCPa and FCPb from the diatom Cyclotella meneghiniana was investigated in a comprehensive study using transient absorption in the visible and near infrared spectral region as well as static fluorescence spectroscopy. The altered oligomerization state of both antenna systems results in a more efficient energy transfer for FCPa, which is also reflected in the different chlorophyll a fluorescence quantum yields. We therefore assume an increased quenching in the higher oligomers of FCPb. The influence of the carotenoid composition was investigated using FCPa and FCPb samples grown under different light conditions and excitation wavelengths at the blue (500 nm) and red (550 nm) wings of the carotenoid absorption. The different light conditions yield in altered amounts of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Since no significant dynamic changes are observed for high light and low light samples, the contribution of the xanthophyll cycle pigments to the energy transfer is most likely negligible. On the contrary, the observed dynamics change drastically for the different excitation wavelengths. The analyses of the decay associated spectra of FCPb suggest an altered energy transfer pathway. For FCPa even an additional time constant was found after excitation at 500 nm. It is assigned to the intrinsic lifetime of either the xanthophyll cycle carotenoids or more probable the blue absorbing fucoxanthins. Based on our studies we propose a detailed model explaining the different excitation energy transfer pathways in FCPa.     

Disentangling two non-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77 K

Diatoms, which are primary producers in the oceans, can rapidly switch on/off efficient photoprotection to respond to fast light-intensity changes in moving waters. The corresponding thermal dissipation of excess-absorbed-light energy can be observed as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Fluorescence-induction measurements on Cyclotella meneghiniana diatoms show two NPQ processes: qE₁ relaxes rapidly in the dark while qE₂ remains present upon switching to darkness and is related to the presence of the xanthophyll-cycle pigment diatoxanthin (Dtx). We performed picosecond fluorescence measurements on cells locked in different (quenching) states, revealing the following sequence of events during full development of NPQ. At first, trimers of light-harvesting complexes (fucoxanthin–chlorophyll a/c proteins), or FCPa, become quenched, while being part of photosystem II (PSII), due to the induced pH gradient across the thylakoid membrane. This is followed by (partial) detachment of FCPa from PSII after which quenching persists. The pH gradient also causes the formation of Dtx which leads to further quenching of isolated PSII cores and some aggregated FCPa. In subsequent darkness, the pH gradient disappears but Dtx remains present and quenching partly pertains. Only in the presence of some light the system completely recovers to the unquenched state.     

Ultrafast multi-pulse transient absorption spectroscopy of fucoxanthin chlorophyll a protein from Phaeodactylum tricornutum

We have applied femtosecond transient absorption spectroscopy in pump-probe and pump-dump-probe regimes to study energy transfer between fucoxanthin and Chl a in fucoxanthin-Chl a complex from the pennate diatom Phaeodactylum tricornutum. Experiments were carried out at room temperature and 77?K to reveal temperature dependence of energy transfer. At both temperatures, the ultrafast (<100?fs) energy transfer channel from the fucoxanthin S₂ state is active and is complemented by the second pathway via the combined S₁/ICT state. The S₁/ICT-Chl a pathway has two channels, the fast one characterized by sub-picosecond energy transfer, and slow having time constants of 4.5?ps at room temperature and 6.6?ps at 77?K. The overall energy transfer via the S₁/ICT is faster at 77?K, because the fast component gains amplitude upon lowering the temperature. The pump-dump-probe regime, with the dump pulse centered in the spectral region of ICT stimulated emission at 950?nm and applied at 2?ps after excitation, proved that the S₁ and ICT states of fucoxanthin in FCP are individual, yet coupled entities. Analysis of the pump-dump-probe data suggested that the main energy donor in the slow S₁/ICT-Chl a route is the S₁ part of the S₁/ICT potential surface.     

Cation-dependent changes in the thylakoid membrane appression of the diatom Thalassiosira pseudonana

Diatoms show a special organisation of their plastid membranes, such that their thylakoids span the entire plastid in bands of three. While in higher plants the interaction of the light harvesting complex II and photosystem II with divalent cations (especially Mg²⁺) was found to take part in the interplay of electrostatic attraction and repulsion in grana membrane appression, for diatoms the key players in maintaining proper membrane distances were not identified so far. In this work, we investigated the changes in the thylakoid architecture of Thalassiosira pseudonana in reaction to different salts by using circular dichroism and fluorescence spectroscopy in combination with other techniques. We show that divalent cations have an important influence on optimal pigment organisation and thus also on maintaining membrane appression. Thereby, monovalent cations are far less effective. The concentration needed is in a physiological range and fits well with the values obtained for higher plant grana stacking, despite the fact that strict protein segregation as seen in higher plant grana is missing.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophylla/c light-harvesting complex in the diatomPhaeodactylum tricornutum

Summary iserum against two polypeptides of the major fucoxanthin-chlorophylla/c light-harvesting complex of the diatomPhaeodactylum tricornutum and heterologous antiserum against purified photosystem I particles of maize were used to localize these two complexes on the thylakoid membranes ofP. tricornutum. As in many chromophyte algae, the thylakoids are loosely appressed and organized into extended bands of three, giving a ratio of 21 for appressed versus non-appressed membranes. Immunoelectron microscopy demonstrated that the fucoxanthin-chlorophylla/c light-harvesting complex, which is believed to be associated with photosystem II, was equally distributed on the appressed and non-appressed thylakoid membranes. Photosystem I was also found on both types of membranes, but was slightly more concentrated on the two outer non-appressed membranes of each band. Similarly, photosystem I activity, as measured by the photooxidation of 3,3-diaminobenzidine, was higher in the outer thylakoids than in the central thylakoid of each band. We conclude that the thylakoids of diatoms differ from those of green algae and higher plants in their macromolecular organization as well as in their morphological arrangement.Abbreviations BSA bovine serum albumin - DAB 3,3-diaminobenzidine - FCPC fucoxanthin-chlorophylla/c light-harvesting complex - LHC light-harvesting complex - PBS phosphate-buffered saline - PS photosystem     

Energy transfer dynamics in a red-shifted violaxanthin-chlorophyll a light-harvesting complex

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690?nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S₂ and hot S₁ states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.     

Energy transfer in the B800-850-carotenoid light-harvesting complex of various mutants of Rhodopseudomonas sphaeroides and of Rhodopseudomonas capsulata

Emission and absorption spectra in the temperature range 4–300 K have been obtained for bacteriochlorophyll light-harvesting complexes (B800–850 complexes) from several mutants of Rhodopseudomonas sphaeroides and a nonphotosynthetic mutant of Rhodopseudomonas capsulata. The energy-transfer properties of these complexes were remarkably similar despite differences in carotenoid composition. Between 300 and 200 K the excitation densities in B800 and B850 are in thermal equilibrium, indicating rapid energy transfer from B800 to B850 and vice versa. The temperature dependence of the ratio of the B800 and B850 emission yields allows the determination of the ratio of the number of B800 and B850 molecules in the complex which is close to 0.5. Below 200 K thermal equilibrium no longer exists. At 4–100 K the B800 emission yield increases with decreasing temperature and becomes dependent on the wavelength of excitation. From the B800 emission yield at 4 K the B800–850 dipole-dipole distance was calculated to be equal to or smaller than 21 Å for all B800–850 complexes. Excitation spectra for B800 and B850 emission show that the overall energy-transfer efficiencies from carotenoid and B800 to B850 are greater than 90% at all temperatures. At 4 K the carotenoid transfers its excitation energy preferentially to B850. Experiments with chromatophores indicated that the energy-transfer properties of the B800–850 complexes were not modified by the isolation procedures.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

The role of far-red spectral states in the energy regulation of phycobilisomes

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.     

Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2.5 A resolution

The plant light-harvesting complex of photosystem II (LHC-II) collects and transmits solar energy for photosynthesis in chloroplast membranes and has essential roles in regulation of photosynthesis and in photoprotection. The 2.5 A structure of pea LHC-II determined by X-ray crystallography of stacked two-dimensional crystals shows how membranes interact to form chloroplast grana, and reveals the mutual arrangement of 42 chlorophylls a and b, 12 carotenoids and six lipids in the LHC-II trimer. Spectral assignment of individual chlorophylls indicates the flow of energy in the complex and the mechanism of photoprotection in two close chlorophyll a-lutein pairs. We propose a simple mechanism for the xanthophyll-related, slow component of nonphotochemical quenching in LHC-II, by which excess energy is transferred to a zeaxanthin replacing violaxanthin in its binding site, and dissipated as heat. Our structure shows the complex in a quenched state, which may be relevant for the rapid, pH-induced component of nonphotochemical quenching.     

Acid denaturation of the B875 light-harvesting complex in membranes of Rhodobacter sphaeroides

The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800–850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a free pigment band; the remaining absorption appeared at 880 nm and shifted to 845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min^-1 for the initial rapid change and 0.05 and 0.11 min^-1 for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.Abbreviations BChl bacteriochlorophyll a - BPheo bacteriopheophytin a     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic -helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.This study is dedicated to Prof. Dr. W Rüdiger on the occasion of his 60th birthday