The Carbonyl Group in ß2 of the Carotenoid Tunes the Photocycle Kinetics in Orange Carotenoid Protein.

Adaptation to rapid environmental changes is crucial for maintaining optimal photosynthetic efficiency and is ultimately key to the survival of all photosynthetic organisms. Like most of them, cyanobacteria protect their photosynthetic apparatus against rapidly increasing light intensities by nonphotochemical quenching (NPQ). In cyanobacteria, NPQ is controlled by Orange Carotenoid Protein (OCP) photocycle. OCP is the only known photoreceptor that uses carotenoid for its light activation. How carotenoid drives and controls this unique photoactivation process is still unknown. However, understanding and potentially controlling the OCP photocycle may open up new possibilities for improving photosynthetic biomass. Here we investigate the effect of the carbonyl group in the ß2 ring of the carotenoid on the OCP photocycle. We report microsecond to minute OCP light activation kinetics and Arrhenius plots of the two OCP forms: Canthaxanthin-bound OCP (OCPCAN) and echinenone-bound OCP (OCPECH). The difference between the two carotenoids is the presence of a carbonyl group in the ß2-ring located in the N-terminal domain of the protein. A combination of temperature-dependent spectroscopy, flash photolysis, and pump-probe transient absorption allows us to report the previously unresolved OCP intermediate associated primarily with the absorption bleach (OCPB). OCPB dominates the photokinetics in the µs to subms time range for OCPCAN and in the µs to ms range for OCPECH. We show that in OCPCAN the OCP photocycle steps are always faster than in OCPECH: from 2 to almost 20 times depending on the step. These results suggest that the presence of the carbonyl group in the ß2-ring of the carotenoid accelerates the OCP photocycle.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Light activation of Orange Carotenoid Protein reveals bicycle-pedal single-bond isomerization.

Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9'-C8' and C7'-C6' single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.

Photosynthesis without ß-carotene.

Carotenoids are essential in oxygenic photosynthesis: they stabilize the pigment-protein complexes, are active in harvesting sunlight and in photoprotection. In plants, they are present as carotenes and their oxygenated derivatives, xanthophylls. While mutant plants lacking xanthophylls are capable of photoautotrophic growth, no plants without carotenes in their photosystems have been reported so far, which has led to the common opinion that carotenes are essential for photosynthesis. Here, we report the first plant that grows photoautotrophically in the absence of carotenes: a tobacco plant containing only the xanthophyll astaxanthin. Surprisingly, both photosystems are fully functional despite their carotenoid-binding sites being occupied by astaxanthin instead of ß-carotene or remaining empty (i.e. are not occupied by carotenoids). These plants display non-photochemical quenching, despite the absence of both zeaxanthin and lutein and show that tobacco can regulate the ratio between the two photosystems in a very large dynamic range to optimize electron transport.

Most life on Earth depends on photosynthesis, the process used by plants and many other organisms to store energy from sunlight and produce oxygen. The first steps of photosynthesis, the capture and conversion of sunlight into chemical energy, happen in large assemblies of proteins containing many pigment molecules called photosystems. In plants, the pigments involved in photosynthesis are green chlorophylls and carotenoids. In addition to harvesting light, carotenoids have an important role in preventing damage caused by overexposure to sunlight There are over one thousand different carotenoids in living beings, but only one, ß-carotene, is present in every organism that performs the type of photosynthesis in which oxygen is released, and is thought to be essential for the process. However, this could never be proved because it is impossible to remove ß-carotene from cells using typical genetic approaches without affecting all other carotenoids. Xu et al. used genetic engineering to create tobacco plants that produced a pigment called astaxanthin in place of ß-carotene. Astaxanthin is a carotenoid from salmon and shrimp, not normally found in plants. These plants are the first living things known to perform photosynthesis without ß-carotene and demonstrate that this pigment is not essential for photosynthesis as long as other carotenoids are present. Xu et al. also show that the photosystems can adapt to using different carotenoids, and can even operate with a reduced number of them. Xu et al's findings show the high flexibility of photosynthesis in plants, which are able to incorporate non-native elements to the process. These results are also important in the context of increasing the photosynthetic efficiency, and thus the productivity of crops, since they show that a radical redesign of the photosynthetic machinery is feasible.

Light-harvesting complex II is an antenna of photosystem I in dark-adapted plants.

Photosystem I (PSI) is a major player in the light reactions of photosynthesis. In higher plants, it consists of a core complex and four external antennae, Lhca1-4 forming the PSI-light-harvesting complex I (LHCI) supercomplex. The protein and pigment composition as well as the spectroscopic properties of this complex are considered to be identical in different higher plant species. In addition to the four Lhca, a pool of mobile LHCII increases the antenna size of PSI under most light conditions. In this work, we have first investigated purified PSI complexes and then PSI in vivo upon long-term dark-adaptation of four well-studied plant species: Arabidopsis thaliana, Zea mays, Nicotiana tabacum and Hordeum vulgare. By performing time-resolved fluorescence measurements, we show that LHCII is associated with PSI also in a dark-adapted state in all the plant species investigated. The number of LHCII subunits per PSI is plant-dependent, varying between one and three. Furthermore, we show that the spectroscopic properties of PSI-LHCI supercomplexes differ in different plants.

Light Acclimation of the Colonial Green Alga Botryococcus braunii Strain Showa.

In contrast to single cellular species, detailed information is lacking on the processes of photosynthetic acclimation for colonial algae, although these algae are important for biofuel production, ecosystem biodiversity, and wastewater treatment. To investigate differences between single cellular and colonial species, we studied the regulation of photosynthesis and photoprotection during photoacclimation for the colonial green alga Botryococcus braunii and made a comparison with the properties of the single cellular species Chlamydomonas reinhardtii We show that B. braunii shares some high-light (HL) photoacclimation strategies with C. reinhardtii and other frequently studied green algae: decreased chlorophyll content, increased free carotenoid content, and increased nonphotochemical quenching (NPQ). Additionally, B. braunii has unique HL photoacclimation strategies, related to its colonial form: strong internal shading by an increase of the colony size and the accumulation of extracellular echinenone (a ketocarotenoid). HL colonies are larger and more spatially heterogenous than low-light colonies. Compared with surface cells, cells deeper inside the colony have increased pigmentation and larger photosystem II antenna size. The core of the largest of the HL colonies does not contain living cells. In contrast with C. reinhardtii, but similar to other biofilm-forming algae, NPQ capacity is substantial in low light. In HL, NPQ amplitude increases, but kinetics are unchanged. We discuss possible causes of the different acclimation responses of C. reinhardtii and B. braunii Knowledge of the specific photoacclimation processes for this colonial green alga further extends the view of the diversity of photoacclimation strategies in photosynthetic organisms.

Time-resolved fluorescence measurements on leaves: principles and recent developments.

Photosynthesis starts when a pigment in the photosynthetic antennae absorbs a photon. The electronic excitation energy is then transferred through the network of light-harvesting pigments to special chlorophyll (Chl) molecules in the reaction centres, where electron transfer is initiated. Energy transfer and primary electron transfer processes take place on timescales ranging from femtoseconds to nanoseconds, and can be monitored in real time via time-resolved fluorescence spectroscopy. This method is widely used for measurements on unicellular photosynthetic organisms, isolated photosynthetic membranes, and individual complexes. Measurements on intact leaves remain a challenge due to their high structural heterogeneity, high scattering, and high optical density, which can lead to optical artefacts. However, detailed information on the dynamics of these early steps, and the underlying structure-function relationships, is highly informative and urgently required in order to get deeper insights into the physiological regulation mechanisms of primary photosynthesis. Here, we describe a current methodology of time-resolved fluorescence measurements on intact leaves in the picosecond to nanosecond time range. Principles of fluorescence measurements on intact leaves, possible sources of alterations of fluorescence kinetics and the ways to overcome them are addressed. We also describe how our understanding of the organisation and function of photosynthetic proteins and energy flow dynamics in intact leaves can be enriched through the application of time-resolved fluorescence spectroscopy on leaves. For that, an example of a measurement on Zea mays leaves is presented.

Dynamic Changes between Two LHCX-Related Energy Quenching Sites Control Diatom Photoacclimation.

Marine diatoms are prominent phytoplankton organisms that perform photosynthesis in extremely variable environments. Diatoms possess a strong ability to dissipate excess absorbed energy as heat via nonphotochemical quenching (NPQ). This process relies on changes in carotenoid pigment composition (xanthophyll cycle) and on specific members of the light-harvesting complex family specialized in photoprotection (LHCXs), which potentially act as NPQ effectors. However, the link between light stress, NPQ, and the existence of different LHCX isoforms is not understood in these organisms. Using picosecond fluorescence analysis, we observed two types of NPQ in the pennate diatom Phaeodactylum tricornutum that were dependent on light conditions. Short exposure of low-light-acclimated cells to high light triggers the onset of energy quenching close to the core of photosystem II, while prolonged light stress activates NPQ in the antenna. Biochemical analysis indicated a link between the changes in the NPQ site/mechanism and the induction of different LHCX isoforms, which accumulate either in the antenna complexes or in the core complex. By comparing the responses of wild-type cells and transgenic lines with a reduced expression of the major LHCX isoform, LHCX1, we conclude that core complex-associated NPQ is more effective in photoprotection than is the antenna complex. Overall, our data clarify the complex molecular scenario of light responses in diatoms and provide a rationale for the existence of a degenerate family of LHCX proteins in these algae.

Photoprotection strategies of the alga Nannochloropsis gaditana.

Nannochloropsis spp. are algae with high potential for biotechnological applications due to their capacity to accumulate lipids. However, little is known about their photosynthetic apparatus and acclimation/photoprotective strategies. In this work, we studied the mechanisms of non-photochemical quenching (NPQ), the fast response to high light stress, in Nannochloropsis gaditana by "locking" the cells in six different states during quenching activation and relaxation. Combining biochemical analysis with time-resolved fluorescence spectroscopy, we correlated each NPQ state with the presence of two well-known NPQ components: de-epoxidized xanthophylls and stress-related antenna proteins (LHCXs). We demonstrated that after exposure to strong light, the rapid quenching that takes place in the antennas of both photosystems was associated with the presence of LHCXs. At later stages, quenching occurs mainly in the antennas of PSII and correlates with the amount of de-epoxidised xanthophylls. We also observed changes in the distribution of excitation energy between photosystems, which suggests redistribution of excitation between photosystems as part of the photo-protective strategy. A multistep model for NPQ induction and relaxation in N. gaditana is discussed.

Zeaxanthin-dependent nonphotochemical quenching does not occur in photosystem I in the higher plant Arabidopsis thaliana.

Nonphotochemical quenching (NPQ) is the process that protects the photosynthetic apparatus of plants and algae from photodamage by dissipating as heat the energy absorbed in excess. Studies on NPQ have almost exclusively focused on photosystem II (PSII), as it was believed that NPQ does not occur in photosystem I (PSI). Recently, Ballottari et al. [Ballottari M, et al. (2014) Proc Natl Acad Sci USA 111:E2431-E2438], analyzing PSI particles isolated from an Arabidopsis thaliana mutant that accumulates zeaxanthin constitutively, have reported that this xanthophyll can efficiently induce chlorophyll fluorescence quenching in PSI. In this work, we have checked the biological relevance of this finding by analyzing WT plants under high-light stress conditions. By performing time-resolved fluorescence measurements on PSI isolated from Arabidopsis thaliana WT in dark-adapted and high-light-stressed (NPQ) states, we find that the fluorescence kinetics of both PSI are nearly identical. To validate this result in vivo, we have measured the kinetics of PSI directly on leaves in unquenched and NPQ states; again, no differences were observed. It is concluded that PSI does not undergo NPQ in biologically relevant conditions in Arabidopsis thaliana The possible role of zeaxanthin in PSI photoprotection is discussed.

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

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.