Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins

In response to iron deficiency, cyanobacteria synthesize the iron stress-induced chlorophyll binding protein IsiA. This protein protects cyanobacterial cells against iron stress. It has been proposed that the protective role of IsiA is related to a blue light-induced nonphotochemical fluorescence quenching (NPQ) mechanism. In iron-replete cyanobacterial cell cultures, strong blue light is known to induce a mechanism that dissipates excess absorbed energy in the phycobilisome, the extramembranal antenna of cyanobacteria. In this photoprotective mechanism, the soluble Orange Carotenoid Protein (OCP) plays an essential role. Here, we demonstrate that in iron-starved cells, blue light is unable to quench fluorescence in the absence of the phycobilisomes or the OCP. By contrast, the absence of IsiA does not affect the induction of fluorescence quenching or its recovery. We conclude that in cyanobacteria grown under iron starvation conditions, the blue light-induced nonphotochemical quenching involves the phycobilisome OCP-related energy dissipation mechanism and not IsiA. IsiA, however, does seem to protect the cells from the stress generated by iron starvation, initially by increasing the size of the photosystem I antenna. Subsequently, the IsiA converts the excess energy absorbed by the phycobilisomes into heat through a mechanism different from the dynamic and reversible light-induced NPQ processes.     

Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures

Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll-carotenoid protein complex known as IsiA or CP43′. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE⁻ mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with - presumably - allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F ₀ and F _m) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F ₀ and F _m) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b ₅₅₉.     

Biogeography of Photosynthetic Light-Harvesting Genes in Marine Phytoplankton

Background

Photosynthetic light-harvesting proteins are the mechanism by which energy enters the marine ecosystem. The dominant prokaryotic photoautotrophs are the cyanobacterial genera Prochlorococcus and Synechococcus that are defined by two distinct light-harvesting systems, chlorophyll-bound protein complexes or phycobilin-bound protein complexes, respectively. Here, we use the Global Ocean Sampling (GOS) Project as a unique and powerful tool to analyze the environmental diversity of photosynthetic light-harvesting genes in relation to available metadata including geographical location and physical and chemical environmental parameters.

Methods

All light-harvesting gene fragments and their metadata were obtained from the GOS database, aligned using ClustalX and classified phylogenetically. Each sequence has a name indicative of its geographic location; subsequent biogeographical analysis was performed by correlating light-harvesting gene budgets for each GOS station with surface chlorophyll concentration.

Conclusion/Significance

Using the GOS data, we have mapped the biogeography of light-harvesting genes in marine cyanobacteria on ocean-basin scales and show that an environmental gradient exists in which chlorophyll concentration is correlated to diversity of light-harvesting systems. Three functionally distinct types of light-harvesting genes are defined: (1) the phycobilisome (PBS) genes of Synechococcus; (2) the pcb genes of Prochlorococcus; and (3) the iron-stress-induced (isiA) genes present in some marine Synechococcus. At low chlorophyll concentrations, where nutrients are limited, the Pcb-type light-harvesting system shows greater genetic diversity; whereas at high chlorophyll concentrations, where nutrients are abundant, the PBS-type light-harvesting system shows higher genetic diversity. We interpret this as an environmental selection of specific photosynthetic strategy. Importantly, the unique light-harvesting system isiA is found in the iron-limited, high-nutrient low-chlorophyll region of the equatorial Pacific. This observation demonstrates the ecological importance of isiA genes in enabling marine Synechococcus to acclimate to iron limitation and suggests that the presence of this gene can be a natural biomarker for iron limitation in oceanic environments.     

Induction of efficient energy dissipation in the isolated light-harvesting complex of Photosystem II in the absence of protein aggregation

Under excess illumination, the Photosystem II light-harvesting antenna of higher plants has the ability to switch into an efficient photoprotective mode, allowing safe dissipation of excitation energy into heat. In this study, we show induction of the energy dissipation state, monitored by chlorophyll fluorescence quenching, in the isolated major light-harvesting complex (LHCII) incorporated into a solid gel system. Removal of detergent caused strong fluorescence quenching, which was totally reversible. Singlet-singlet annihilation and gel electrophoresis experiments suggested that the quenched complexes were in the trimeric not aggregated state. Both the formation and recovery of this quenching state were inhibited by a cross-linker, implying involvement of conformational changes. Absorption and CD measurements performed on the samples in the quenched state revealed specific alterations in the spectral bands assigned to the red forms of chlorophyll a, neoxanthin, and lutein 1 molecules. The majority of these alterations were similar to those observed during LHCII aggregation. This suggests that not the aggregation process as such but rather an intrinsic conformational transition in the complex is responsible for establishment of quenching. 77 K fluorescence measurements showed red-shifted chlorophyll a fluorescence in the 690-705 nm region, previously observed in aggregated LHCII. The fact that all spectral changes associated with the dissipative mode observed in the gel were different from those of the partially denatured complex strongly argues against the involvement of protein denaturation in the observed quenching. The implications of these findings for proposed mechanisms of energy dissipation in the Photosystem II antenna are discussed.     

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F_o) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F_o quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.     

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.     

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     

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA)

The chlorophyll-protein CP43′ (isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the ‘energy-dependent non-photochemical quenching’ (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence.     

Effects of Exogenous Putrescine on Chlorophyll Fluorescence Imaging and Heat Dissipation Capacity in Cucumber (Cucumis sativus L.) Under Salt Stress

The objective of this study was to identify the effects of exogenous putrescine on photosynthetic performance and heat dissipation capacity in cucumber seedlings under salt stress. The stress of 75 mM NaCl for 7 days caused a significant decrease in net photosynthetic rate (P _N ). The experiment employed a chlorophyll fluorescence imaging technique and demonstrated that the maximal quantum yield of photosystem II photochemistry (Fv/Fm) and the actual photochemical efficiency of photosystem II (ΦPSII) were reduced by salt stress. Moreover, salt stress markedly reduced the photochemical quenching coefficient (qP) and non-photochemical quenching coefficient (qN), and significantly increased non-regulated heat dissipation (ΦNO). However, stressed plants supplied with exogenous putrescine exhibited higher P _N and ΦPSII, which indicated that putrescine can alleviate the detrimental effects on photosynthesis induced by salt stress. Putrescine sprayed on stressed plants significantly enhanced the regulated energy dissipation (ΦNPQ) and decreased ΦNO. Application of exogenous putrescine also changed the levels of xanthophyll cycle components and further enhanced the de-epoxidation state of xanthophyll cycle pigments under salt stress. Under control conditions, putrescine exerted little influence on the photosynthetic parameters in cucumber leaves. In conclusion, the application of exogenous putrescine may improve the heat dissipation capacity by promoting the xanthophyll cycle to reduce the damage caused by excess excitation energy, thus enhancing the salt tolerance of cucumber seedlings.     

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

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

Introduction of cysteine-mediated quenching in the CP43 protein of photosystem II builds resilience to high-light stress in a cyanobacterium

Photosystem (PS) II is prone to photodamage both as a direct consequence of light, and indirectly by producing reactive oxygen species. Engineering high-light tolerance in cyanobacteria with minimal impact on PSII function is desirable in synthetic biology. IsiA, a CP43 homolog found exclusively in cyanobacteria, can dissipate excess light energy. We have recently determined that the sole cysteine residue of IsiA in Synechocystis sp. PCC 6803 has a critical role in non-photochemical quenching. Similar cysteine-mediated energy quenching has also been observed in green?sulfur bacteria. Sequence analysis of IsiA and CP43 aligns cysteine 260 of IsiA with valine 277 of CP43 in Synechocystis sp. PCC 6803. In the current study, we explore the impact of replacing valine 277 of CP43 to a cysteine on growth, PSII activity and high-light tolerance. Our results imply a decline in the PSII output for the mutant (CP43V277C) presumably due to the dissipation of absorbed light energy by cysteine. Spectroscopic analysis of isolated PSII from this mutant strain also suggests a delayed transfer of excitation energy from CP43-associated chlorophyll a to PSII reaction center. The mutation makes the PSII high-light tolerant and provides a small advantage in growth under high-light conditions. This previously unexplored strategy to engineer high-light tolerance could be a step further towards developing cyanobacterial cells as biofactories.     

Diurnal changes in photochemical efficiency,the reduction state of Q,radiationless energy dissipation,and non-photochemical fluorescence quenching in cacti exposed to natural sunlight in northern Venezuela

Summary Diurnal measurements of chlorophyll a fluorescence from cacti (Nopalea cochenillifera, Opuntia ficus-indica, and Opuntia wentiana) growing in northern Venezuela were used to determine photochemical fluorescence quenching related to the reduction state of the primary electron acceptor of PS II as well as non-photochemical fluorescence quenching which reflects the fraction of energy going primarily into radiationless deexcitation. The cladodes used in this study were oriented such that one surface received direct sunlight in the morning and the other one during the afternoon. Both surfaces exhibited large increases in radiationless energy dissipation from the photochemical system accompanied by decreases in PS II photochemical efficiency during direct exposure to natural sunlight. During exposure to sunlight in the morning, dissipation of absorbed light energy through photosynthesis and radiationless energy dissipation was sufficient to maintain Q, the primary electron acceptor for PS II, in a low reduction state. During exposure to sunlight in the afternoon, however, the reduction state of Q rose to levels greater than 50%, presumably due to a decrease in photosynthetic electron transport as the decarboxylation of the nocturnally accumulated malic acid was completed. Exposure to direct sunlight in the afternoon also led to more sustained increases in radiationless energy dissipation. Furthermore, the increases in radiationless energy dissipation during exposure of a water-stressed cladode of O. wentiana to direct sunlight were much greater than those from other well-watered cacti, presumably due to sustained stomatal closure and decreased rates of photosynthetic electron transport. These results indicate that the radiationless dissipation of absorbed light is an important process in these CAM plants under natural conditions, and may reflect a protective mechanism against the potentially damaging effects of the accumulation of excessive energy, particularly under conditions where CO₂ availability is restricted.Abbreviations CAM crassulacean acid metabolism - F _o instantaneous fluorescence emission - F _M maximum fluorescence emission - F _v variable fluorescence emission - K _D rate constant for radiationless energy dissipation in the antenna chlorophyll - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q primary electron acceptor of photosystem II - q _NP non-photochemical fluorescence quenching - q _P photochemical fluorescence quenching - T _C cladode temperature     

The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.     

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems’ activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30-40% for both photoreactions—P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core-membrane linker allophycocyanin APC-L_CM is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-L_CM may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE(-) mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with--presumably--allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris

Conifers of the boreal zone encounter considerable combined stress of low temperature and high light during winter, when photosynthetic consumption of excitation energy is blocked. In the evergreen Pinus sylvestris L. these stresses coincided with major seasonal changes in photosystem II (PSII) organisation and pigment composition. The earliest changes occurred in September, before any freezing stress, with initial losses of chlorophyll, the D1-protein of the PSII reaction centre and of PSII light-harvesting-complex (LHC II) proteins. In October there was a transient increase in F₀, resulting from detachment of the light-harvesting antennae as reaction centres lost D1. The D1-protein content eventually decreased to 90%, reaching a minimum by December, but PSII photochemical efficiency [variable fluorescence (F_v)/maximum fluorescence (F_m)] did not reach the winter minimum until mid-February. The carotenoid composition varied seasonally with a twofold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreased from 0.9 to 0.1 from October to January. The loss of chlorophyll was complete by October and during winter much of the remaining chlorophyll was reorganised in aggregates of specific polypeptide composition, which apparently efficiently quench excitation energy through non-radiative dissipation. The timing of the autumn and winter changes indicated that xanthophyll de-epoxidation correlates with winter quenching of chlorophyll fluorescence while the drop in photochemical efficiency relates more to loss of D1-protein. In April and May recovery of the photochemistry of PSII, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occurred concomitantly. Indoor recovery of photosynthesis in winter-stressed branches under favourable conditions was completed within 3 d, with rapid increases in F₀, the epoxidation state of the xanthophylls and in light-harvesting polypeptides, followed by recovery of D1-protein content and F_v/F_m, all without net increase in chlorophyll. The fall and winter reorganisation allow Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid recovery of photosynthesis in spring.Abbreviations Elips early light-induced proteins - EPS epoxidation state - F₀ instantaneous fluorescence - F_m maximum fluorescence - F_v variable fluorescence - LHC II light-harvesting complex of PSII - LiDS lithium dodecyl sulfate This research was supported by the Swedish Natural Science Research Council. We wish to thank Dr. Adrian Clarke¹ (Department of Plant Physiology, University of Umeå, Sweden) for advice on electrophoresis, valuable discussion and providing antibodies. Dr. Stefan Jansson¹ and Dr. Torill Hundal (Department for Biochemistry, University of Stockholm, Sweden) provided antibodies. Jan Karlsson¹ helped with the HPLC, Dr. Marianna Krol gave advice on green gels and Dr. Vaughan Hurry (Cooperative Research Centre for Plant Sciences, Australian National University, Canberra, Australia) provided valuable discussion.