Remembering Jan Amesz (1934-2001): a great gentleman, a major discoverer, and an internationally renowned biophysicist of both oxygenic and anoxygenic photosynthesisa.

We present here the research contributions of Jan Amesz (1934-2001) on deciphering the details of the early physico-chemical steps in oxygenic photosynthesis in plants, algae and cyanobacteria, as well as in anoxygenic photosynthesis in purple, green, and heliobacteria. His research included light absorption and the mechanism of excitation energy transfer, primary photochemistry, and electron transfer steps until the reduction of pyridine nucleotides. Among his many discoveries, we emphasize his 1961 proof, with L. N. M. Duysens, of the "series scheme" of oxygenic photosynthesis, through antagonistic effects of Light I and II on the redox state of cytochrome f. Further, we highlight the following research on oxygenic photosynthesis: the experimental direct proof that plastoquinone and plastocyanin function at their respective places in the Z-scheme. In addition, Amesz's major contributions were in unraveling the mechanism of excitation energy transfer and electron transport steps in anoxygenic photosynthetic bacteria (purple, green and heliobacteria). Before we present his research, focusing on his key discoveries, we provide a glimpse of his personal life. We end this Tribute with reminiscences from three of his former doctoral students (Sigi Neerken; Hjalmar Pernentier, and Frank Kleinherenbrink) and from several scientists (Suleyman Allakhverdiev; Robert Blankenship; Richard Cogdell) including two of the authors (G. Garab and A. Stirbet) of this Tribute.

Role of isotropic lipid phase in the fusion of photosystem II membranes.

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

Honoring two stalwarts of photosynthesis research: Eva-Mari Aro and Govindjee.

On behalf of the entire photosynthesis community, it is an honor, for us, to write about two very eminent scientists who were recently recognised with a Lifetime Achievement Award from the International Society of Photosynthesis Research (ISPR) on August 5, 2022; this prestigious Award was given during the closing ceremony of the 18th International Congress on Photosynthesis Research in Dunedin, New Zealand. The awardees were: Professor Eva-Mari Aro (Finland) and Professor Emeritus Govindjee Govindjee (USA). One of the authors, Anjana Jajoo, is especially delighted to be a part of this tribute to professors Aro and Govindjee as she was lucky enough to have worked with both of them.

New foundations for the physical mechanism of variable chlorophyll a fluorescence. Quantum efficiency versus the light-adapted state of photosystem II.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons to fix CO2. Although the structure at atomic resolution and the basic photophysical and photochemical functions of PSII are well understood, many important questions remain. The activity of PSII in vitro and in vivo is routinely monitored by recording the induction kinetics of chlorophyll a fluorescence (ChlF). According to the 'mainstream' model, the rise from the minimum level (Fo) to the maximum (Fm) of ChlF of dark-adapted PSII reflects the closure of all functionally active reaction centers, and the Fv/Fm ratio is equated with the maximum photochemical quantum yield of PSII (where Fv=Fm-Fo). However, this model has never been free of controversies. Recent experimental data from a number of studies have confirmed that the first single-turnover saturating flash (STSF), which generates the closed state (PSIIC), produces F1

Etioplasts are more susceptible to salinity stress than chloroplasts and photosynthetically active etio-chloroplasts of wheat (Triticum aestivum L.).

High soil salinity is a global problem in agriculture that directly affects seed germination and the development of the seedlings sown deep in the soil. To study how salinity affected plastid ultrastructure, leaf segments of 11-day-old light- and dark-grown (etiolated) wheat (Triticum aestivum L. cv. Mv Béres) seedlings were floated on Hoagland solution, 600 mM KCl:NaCl (1:1) salt or isosmotic polyethylene glycol solution for 4 h in the dark. Light-grown seedlings were also treated in the light. The same treatments were also performed on etio-chloroplasts of etiolated seedlings greened for different time periods. Salt stress induced slight to strong changes in the relative chlorophyll content, photosynthetic activity, and organization of thylakoid complexes. Measurements of malondialdehyde contents and high-temperature thermoluminescence indicated significantly increased oxidative stress and lipid peroxidation under salt treatment, except for light-grown leaves treated in the dark. In chloroplasts of leaf segments treated in the light, slight shrinkage of grana (determined by transmission electron microscopy and small-angle neutron scattering) was observed, while a swelling of the (pro)thylakoid lumen was observed in etioplasts. Salt-induced swelling disappeared after the onset of photosynthesis after 4 h of greening. Osmotic stress caused no significant alterations in plastid structure and only mild changes in their activities, indicating that the swelling of the (pro)thylakoid lumen and the physiological effects of salinity are rather associated with the ionic component of salt stress. Our data indicate that etioplasts of dark-germinated wheat seedlings are the most sensitive to salt stress, especially at the early stages of their greening.

Ultrafast excitation quenching by the oxidized photosystem II reaction center.

Photosystem II (PSII) is the pigment-protein complex driving the photoinduced oxidation of water and reduction of plastoquinone in all oxygenic photosynthetic organisms. Excitations in the antenna chlorophylls are photochemically trapped in the reaction center (RC) producing the chlorophyll-pheophytin radical ion pair P+ Pheo-. When electron donation from water is inhibited, the oxidized RC chlorophyll P+ acts as an excitation quencher, but knowledge on the kinetics of quenching is limited. Here, we used femtosecond transient absorption spectroscopy to compare the excitation dynamics of PSII with neutral and oxidized RC (P+). We find that equilibration in the core antenna has a major lifetime of about 300 fs, irrespective of the RC redox state. Two-dimensional electronic spectroscopy revealed additional slower energy equilibration occurring on timescales of 3-5 ps, concurrent with excitation trapping. The kinetics of PSII with open RC can be described well with previously proposed models according to which the radical pair P+ Pheo- is populated with a main lifetime of about 40 ps, which is primarily determined by energy transfer between the core antenna and the RC chlorophylls. Yet, in PSII with oxidized RC (P+), fast excitation quenching was observed with decay lifetimes as short as 3 ps and an average decay lifetime of about 90 ps, which is shorter than the excited-state lifetime of PSII with open RC. The underlying mechanism of this extremely fast quenching prompts further investigation.

Characterization of the Rate-Limiting Steps in the Dark-To-Light Transitions of Closed Photosystem II: Temperature Dependence and Invariance of Waiting Times during Multiple Light Reactions.

Rate-limiting steps in the dark-to-light transition of Photosystem II (PSII) were discovered by measuring the variable chlorophyll-a fluorescence transients elicited by single-turnover saturating flashes (STSFs). It was shown that in diuron-treated samples: (i) the first STSF, despite fully reducing the QA quinone acceptor molecule, generated only an F1(

Neutron scattering in photosynthesis research: recent advances and perspectives for testing crop plants.

The photosynthetic performance of crop plants under a variety of environmental factors and stress conditions, at the fundamental level, depends largely on the organization and structural flexibility of thylakoid membranes. These highly organized membranes accommodate virtually all protein complexes and additional compounds carrying out the light reactions of photosynthesis. Most regulatory mechanisms fine-tuning the photosynthetic functions affect the organization of thylakoid membranes at different levels of the structural complexity. In order to monitor these reorganizations, non-invasive techniques are of special value. On the mesoscopic scale, small-angle neutron scattering (SANS) has been shown to deliver statistically and spatially averaged information on the periodic organization of the thylakoid membranes in vivo and/or, in isolated thylakoids, under physiologically relevant conditions, without fixation or staining. More importantly, SANS investigations have revealed rapid reversible reorganizations on the timescale of several seconds and minutes. In this paper, we give a short introduction into the basics of SANS technique, advantages and limitations, and briefly overview recent advances and potential applications of this technique in the physiology and biotechnology of crop plants. We also discuss future perspectives of neutron crystallography and different neutron scattering techniques, which are anticipated to become more accessible and of more use in photosynthesis research at new facilities with higher fluxes and innovative instrumentation.

Accumulation of geranylgeranylated chlorophylls in the pigment-protein complexes of Arabidopsis thaliana acclimated to green light: effects on the organization of light-harvesting complex II and photosystem II functions.

Light quality significantly influences plant metabolism, growth and development. Recently, we have demonstrated that leaves of barley and other plant species grown under monochromatic green light (500-590 nm) accumulated a large pool of chlorophyll a (Chl a) intermediates with incomplete hydrogenation of their phytyl chains. In this work, we studied accumulation of these geranylgeranylated Chls a and b in pigment-protein complexes (PPCs) of Arabidopsis plants acclimated to green light and their structural-functional consequences on the photosynthetic apparatus. We found that geranylgeranylated Chls are present in all major PPCs, although their presence was more pronounced in light-harvesting complex II (LHCII) and less prominent in supercomplexes of photosystem II (PSII). Accumulation of geranylgeranylated Chls hampered the formation of PSII and PSI super- and megacomplexes in the thylakoid membranes as well as their assembly into chiral macrodomains; it also lowered the temperature stability of the PPCs, especially that of LHCII trimers, which led to their monomerization and an anomaly in the photoprotective mechanism of non-photochemical quenching. Role of geranylgeranylated Chls in adverse effects on photosynthetic apparatus of plants acclimated to green light is discussed.

Bee Venom Melittin Disintegrates the Respiration of Mitochondria in Healthy Cells and Lymphoblasts, and Induces the Formation of Non-Bilayer Structures in Model Inner Mitochondrial Membranes.

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O2 consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O2 consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin's cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by 31P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane.

Differential Polarization Imaging of Plant Cells. Mapping the Anisotropy of Cell Walls and Chloroplasts.

Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general-in addition to their high sensitivity, fast data acquisition, and great versatility of 2D-4D image analyses-also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments-making them capable of measuring different polarization spectroscopy parameters, parallel with the 'conventional' intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies.

Changes in aggregation states of light-harvesting complexes as a mechanism for modulating energy transfer in desert crust cyanobacteria.

In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.

Effects of selenate and red Se-nanoparticles on the photosynthetic apparatus of Nicotiana tabacum.

Selenium (Se) is a natural trace element, which shifts its action in a relatively narrow concentration range from nutritional role to toxicity. Although it has been well established that in plants chloroplasts are among the primary targets, the mechanism of toxicity on photosynthesis is not well understood. Here, we compared selenate and red-allotrope elemental selenium nanoparticles (red nanoSe) in in vitro tobacco cultures to investigate their effects on the structure and functions of the photosynthetic machinery. Selenate at 10 mg/L concentration retarded plant growth; it also led to a decreased chlorophyll content, accompanied with an increase in the carotenoid-to-chlorophyll ratio. Structural examinations of the photosynthetic machinery, using electron microscopy, small-angle neutron scattering and circular dichroism spectroscopy, revealed significant perturbation in the macro-organization of the pigment-protein complexes and sizeable shrinkage in the repeat distance of granum thylakoid membranes. As shown by chlorophyll a fluorescence transient measurements, these changes in the ultrastructure were associated with a significantly diminished photosystem II activity and a reduced performance of the photosynthetic electron transport, and an enhanced capability of non-photochemical quenching. These changes in the structure and function of the photosynthetic apparatus explain, at least in part, the retarded growth of plantlets in the presence of 10 mg/L selenate. In contrast, red nanoSe, even at 100 mg/L and selenate at 1 mg/L, exerted no negative effect on the growth of plantlets and affected only marginally the thylakoid membrane ultrastructure and the photosynthetic functions.

Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment.

Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples-without interfering with their 'conventional' fluorescence or transmission imaging (Steinbach et al. in Methods Appl Fluoresc 2:015005, 2014). The resolution and the quality of fluorescence imaging have been enhanced in the recently constructed Re-scan confocal microscopy (RCM) (De Luca et al. in Biomed Opt Express 4:2644-2656, 2013). In this work, we developed the RCM technique further, by adding a DP-attachment modulating the exciting laser beam via a liquid crystal (LC) retarder synchronized with the data acquisition system; by this means, and with the aid of a software, fluorescence-detected linear dichroism (FDLD), characteristic of the anisotropic molecular organization of the sample, could be recorded in parallel with the confocal fluorescence imaging. For demonstration, we show FDLD images of a plant cell wall (Ginkgo biloba) stained with Etzold's staining solution.

Lipid-polymorphism of plant thylakoid membranes. Enhanced non-bilayer lipid phases associated with increased membrane permeability.

Earlier experiments, using 31 P-NMR and time-resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non-bilayer (or non-lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in 31 P-NMR signatures. However, changes in the lipid-phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording 31 P-NMR spectra on isolated thylakoid membranes that were suspended in sorbitol- or NaCl-based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol-based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash-induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a-fluorescence transients; all variations were much less pronounced in the NaCl-based medium. These observations suggest that non-bilayer lipids and non-lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 µs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Imaging linear and circular polarization features in leaves with complete Mueller matrix polarimetry.

Spectropolarimetry of intact plant leaves allows to probe the molecular architecture of vegetation photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological information. In addition to the molecular signals due to the photosynthetic machinery, the cell structure and its arrangement within a leaf can create and modify polarization signals. Using Mueller matrix polarimetry with rotating retarder modulation, we have visualized spatial variations in polarization in transmission around the chlorophyll a absorbance band from 650 nm to 710 nm. We show linear and circular polarization measurements of maple leaves and cultivated maize leaves and discuss the corresponding Mueller matrices and the Mueller matrix decompositions, which show distinct features in diattenuation, polarizance, retardance and depolarization. Importantly, while normal leaf tissue shows a typical split signal with both a negative and a positive peak in the induced fractional circular polarization and circular dichroism, the signals close to the veins only display a negative band. The results are similar to the negative band as reported earlier for single macrodomains. We discuss the possible role of the chloroplast orientation around the veins as a cause of this phenomenon. Systematic artefacts are ruled out as three independent measurements by different instruments gave similar results. These results provide better insight into circular polarization measurements on whole leaves and options for vegetation remote sensing using circular polarization.

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes - As revealed by small-angle neutron scattering.

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H (pD) and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity - similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.

Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes.

Diatoms possess special light-harvesting proteins involved in the photoprotection mechanism called non-photochemical quenching (NPQ). These Lhcx proteins were shown to be subunits of trimeric fucoxanthin-chlorophyll complexes (FCPa) in centric diatoms, but their mode of action is still unclear. Here we investigated the influence of Fcp6, an orthologue to Lhcx1 of Thalassiosira pseudonana in the diatom Cyclotella meneghiniana, by reducing its amount using an antisense approach. Whereas the pigment interactions inside FCPa were not influenced by the presence or absence of Fcp6, as demonstrated by unaltered spectra of circular dichroism, changes could be observed on the level of thylakoids and cells in the mutants compared to WT. This fits to recent models of NPQ in diatoms, where FCP aggregation or supramolecular reorganisation is thought to be a major feature. Thus, Fcp6 (Lhcx1) appears to alter pigment-pigment interactions inside the aggregates, but not inside (un-aggregated) FCPa itself.