Roles for leakiness and O2 evolution in explaining lower-than-theoretical quantum yields of photosynthesis in the PEP-CK subtype of C4 plants.

Theoretically, the PEP-CK C4 subtype has a higher quantum yield of CO2 assimilation ( Φ CO 2 ) than NADP-ME or NAD-ME subtypes because ATP required for operating the CO2-concentrating mechanism is believed to mostly come from the mitochondrial electron transport chain (mETC). However, reported Φ CO 2 is not higher in PEP-CK than in the other subtypes. We hypothesise, more photorespiration, associated with higher leakiness and O2 evolution in bundle-sheath (BS) cells, cancels out energetic advantages in PEP-CK species. Nine species (two to four species per subtype) were evaluated by gas exchange, chlorophyll fluorescence, and two-photon microscopy to estimate the BS conductance (gbs) and leakiness using a biochemical model. Average gbs estimates were 2.9, 4.8, and 5.0 mmol m-2 s-1 bar-1, and leakiness values were 0.129, 0.179, and 0.180, in NADP-ME, NAD-ME, and PEP-CK species, respectively. The BS CO2 level was somewhat higher, O2 level was marginally lower, and thus, photorespiratory loss was slightly lower, in NADP-ME than in NAD-ME and PEP-CK species. Differences in these parameters existed among species within a subtype, and gbs was co-determined by biochemical decarboxylating sites and anatomical characteristics. Our hypothesis and results partially explain variations in observed Φ CO 2 , but suggest that PEP-CK species probably use less ATP from mETC than classically defined PEP-CK mechanisms.

Expansion microscopy resolves the thylakoid structure of spinach.

The light-harvesting reactions of photosynthesis take place on the thylakoid membrane inside chloroplasts. The thylakoid membrane is folded into appressed membranes, the grana, and nonappressed membranes that interconnect the grana, the stroma lamellae. This folding is essential for the correct functioning of photosynthesis. Electron microscopy and atomic force microscopy are commonly used to study the thylakoid membrane, but these techniques have limitations in visualizing a complete chloroplast and its organization. To overcome this limitation, we applied expansion microscopy (ExM) on isolated chloroplasts. ExM is a technique that involves physically expanding a sample in a swellable hydrogel to enhance the spatial resolution of fluorescence microscopy. Using all-protein staining, we visualized the 3D structure of spinach (Spinacia oleracea) thylakoids in detail. We were able to resolve stroma lamellae that were 60 nm apart and observe their helical wrapping around the grana. Furthermore, we accurately measured the dimensions of grana from top views of chloroplasts, which allow for precise determination of the granum diameter. Our results demonstrate that ExM is a fast and reliable technique for studying thylakoid organization in great detail.

The effects of different daily irradiance profiles on Arabidopsis growth, with special attention to the role of PsbS.

In nature, light is never constant, while in the controlled environments used for vertical farming, in vitro propagation, or plant production for scientific research, light intensity is often kept constant during the photoperiod. To investigate the effects on plant growth of varying irradiance during the photoperiod, we grew Arabidopsis thaliana under three irradiance profiles: a square-wave profile, a parabolic profile with gradually increasing and subsequently decreasing irradiance, and a regime comprised of rapid fluctuations in irradiance. The daily integral of irradiance was the same for all three treatments. Leaf area, plant growth rate, and biomass at time of harvest were compared. Plants grown under the parabolic profile had the highest growth rate and biomass. This could be explained by a higher average light-use efficiency for carbon dioxide fixation. Furthermore, we compared the growth of wild type plants with that of the PsbS-deficient mutant npq4. PsbS triggers the fast non-photochemical quenching process (qE) that protects PSII from photodamage during sudden increases in irradiance. Based mainly on field and greenhouse experiments, the current consensus is that npq4 mutants grow more slowly in fluctuating light. However, our data show that this is not the case for several forms of fluctuating light conditions under otherwise identical controlled-climate room conditions.

Spectral diversity of photosystem I from flowering plants.

Photosystem I and II (PSI and PSII) work together to convert solar energy into chemical energy. Whilst a lot of research has been done to unravel variability of PSII fluorescence in response to biotic and abiotic factors, the contribution of PSI to in vivo fluorescence measurements has often been neglected or considered to be constant. Furthermore, little is known about how the absorption and emission properties of PSI from different plant species differ. In this study, we have isolated PSI from five plant species and compared their characteristics using a combination of optical and biochemical techniques. Differences have been identified in the fluorescence emission spectra and at the protein level, whereas the absorption spectra were virtually the same in all cases. In addition, the emission spectrum of PSI depends on temperature over a physiologically relevant range from 280 to 298 K. Combined, our data show a critical comparison of the absorption and emission properties of PSI from various plant species.

Single chloroplast in folio imaging sheds light on photosystem energy redistribution during state transitions.

Oxygenic photosynthesis is driven by light absorption in photosystem I (PSI) and photosystem II (PSII). A balanced excitation pressure between PSI and PSII is required for optimal photosynthetic efficiency. State transitions serve to keep this balance. If PSII is overexcited in plants and green algae, a mobile pool of light-harvesting complex II (LHCII) associates with PSI, increasing its absorption cross-section and restoring the excitation balance. This is called state 2. Upon PSI overexcitation, this LHCII pool moves to PSII, leading to state 1. Whether the association/dissociation of LHCII with the photosystems occurs between thylakoid grana and thylakoid stroma lamellae during state transitions or within the same thylakoid region remains unclear. Furthermore, although state transitions are thought to be accompanied by changes in thylakoid macro-organization, this has never been observed directly in functional leaves. In this work, we used confocal fluorescence lifetime imaging to quantify state transitions in single Arabidopsis (Arabidopsis thaliana) chloroplasts in folio with sub-micrometer spatial resolution. The change in excitation-energy distribution between PSI and PSII was investigated at a range of excitation wavelengths between 475 and 665 nm. For all excitation wavelengths, the PSI/(PSI + PSII) excitation ratio was higher in state 2 than in state 1. We next imaged the local PSI/(PSI + PSII) excitation ratio for single chloroplasts in both states. The data indicated that LHCII indeed migrates between the grana and stroma lamellae during state transitions. Finally, fluorescence intensity images revealed that thylakoid macro-organization is largely unaffected by state transitions. This single chloroplast in folio imaging method will help in understanding how plants adjust their photosynthetic machinery to ever-changing light conditions.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Photosynthetic Light Harvesting and Thylakoid Organization in a CRISPR/Cas9 Arabidopsis Thaliana LHCB1 Knockout Mutant.

Light absorbed by chlorophylls of Photosystems II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is trimeric LHCII. In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of LHCII. In absence of LHCB1, the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss, leading to a pale green phenotype and growth delay. The Photosystem II absorption cross-section was smaller, while the Photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring Photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by lower Photosystem I over Photosystem II reaction center ratio and by the dephosphorylation of LHCII and Photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knockout lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

The role of light-harvesting complex I in excitation energy transfer from LHCII to photosystem I in Arabidopsis.

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP+. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI-LHCI-LHCII supercomplex. The binding site(s) of the "additional" LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that "additional" LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

State transitions and photosystems spatially resolved in individual cells of the cyanobacterium Synechococcus elongatus.

State transitions are a low-light acclimation response through which the excitation of Photosystem I (PSI) and Photosystem II (PSII) is balanced; however, our understanding of this process in cyanobacteria remains poor. Here, picosecond fluorescence kinetics was recorded for the cyanobacterium Synechococcus elongatus using fluorescence lifetime imaging microscopy (FLIM), both upon chlorophyll a and phycobilisome (PBS) excitation. Fluorescence kinetics of single cells obtained using FLIM were compared with those of ensembles of cells obtained with time-resolved fluorescence spectroscopy. The global distribution of PSI and PSII and PBSs was mapped making use of their fluorescence kinetics. Both radial and lateral heterogeneity were found in the distribution of the photosystems. State transitions were studied at the level of single cells. FLIM results show that PSII quenching occurs in all cells, irrespective of their state (I or II). In S. elongatus cells, this quenching is enhanced in State II. Furthermore, the decrease of PSII fluorescence in State II was homogeneous throughout the cells, despite the inhomogeneous PSI/PSII ratio. Finally, some disconnected PBSs were resolved in most State II cells. Taken together our data show that PSI is enriched in the inner thylakoid, while state transitions occur homogeneously throughout the cell.

State transitions in cyanobacteria studied with picosecond fluorescence at room temperature.

Cyanobacteria can rapidly regulate the relative activity of their photosynthetic complexes photosystem I and II (PSI and PSII) in response to changes in the illumination conditions. This process is known as state transitions. If PSI is preferentially excited, they go to state I whereas state II is induced either after preferential excitation of PSII or after dark adaptation. Different underlying mechanisms have been proposed in literature, in particular i) reversible shuttling of the external antenna complexes, the phycobilisomes, between PSI and PSII, ii) reversible spillover of excitation energy from PSII to PSI, iii) a combination of both and, iv) increased excited-state quenching of the PSII core in state II. Here we investigated wild-type and mutant strains of Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 using time-resolved fluorescence spectroscopy at room temperature. Our observations support model iv, meaning that increased excited-state quenching of the PSII core occurs in state II thereby balancing the photochemistry of photosystems I and II.

The relevance of dynamic thylakoid organisation to photosynthetic regulation.

The higher plant chloroplast thylakoid membrane system performs the light-dependent reactions of photosynthesis. These provide the ATP and NADPH required for the fixation of CO2 into biomass by the Calvin-Benson cycle and a range of other metabolic reactions in the stroma. Land plants are frequently challenged by fluctuations in their environment, such as light, nutrient and water availability, which can create a mismatch between the amounts of ATP and NADPH produced and the amounts required by the downstream metabolism. Left unchecked, such imbalances can lead to the production of reactive oxygen species that damage the plant and harm productivity. Fortunately, plants have evolved a complex range of regulatory processes to avoid or minimize such deleterious effects by controlling the efficiency of light harvesting and electron transfer in the thylakoid membrane. Generally the regulation of the light reactions has been studied and conceptualised at the microscopic level of protein-protein and protein-ligand interactions, however in recent years dynamic changes in the thylakoid macrostructure itself have been recognised to play a significant role in regulating light harvesting and electron transfer. Here we review the evidence for the involvement of macrostructural changes in photosynthetic regulation and review the techniques that brought this evidence to light.

Electrochemically Gated Long-Distance Charge Transport in Photosystem†I.

The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic reactions that are coupled through redox mediators, including proteins and small molecules. The use of native and synthetic redox probes is key to understanding charge transport mechanisms and to the design of bioelectronic sensors and solar energy conversion devices. However, redox probes have limited tunability to exchange charge at the desired electrochemical potentials (energy levels) and at different protein sites. Herein, we take advantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi level and nanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI) complexes. Current-distance measurements at different potentiostatic conditions indicate that PSI supports long-distance transport that is electrochemically gated near the redox potential of P700, with current extending farther under hole injection conditions.

Digitonin-sensitive LHCII enlarges the antenna of Photosystem I in stroma lamellae of Arabidopsis thaliana after far-red and blue-light treatment.

Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna.

State transitions in the cyanobacterium Synechococcus elongatus 7942 involve reversible quenching of the photosystem II core.

Cyanobacteria use chlorophyll and phycobiliproteins to harvest light. The resulting excitation energy is delivered to reaction centers (RCs), where photochemistry starts. The relative amounts of excitation energy arriving at the RCs of photosystem I (PSI) and II (PSII) depend on the spectral composition of the light. To balance the excitations in both photosystems, cyanobacteria perform state transitions to equilibrate the excitation energy. They go to state I if PSI is preferentially excited, for example after illumination with blue light (light I), and to state II after illumination with green-orange light (light II) or after dark adaptation. In this study, we performed 77-K time-resolved fluorescence spectroscopy on wild-type Synechococcus elongatus 7942 cells to measure how state transitions affect excitation energy transfer to PSI and PSII in different light conditions and to test the various models that have been proposed in literature. The time-resolved spectra show that the PSII core is quenched in state II and that this is not due to a change in excitation energy transfer from PSII to PSI (spill-over), either direct or indirect via phycobilisomes.

Dynamic feedback of the photosystem II reaction centre on photoprotection in plants.

Photosystem II of higher plants is protected against light damage by thermal dissipation of excess excitation energy, a process that can be monitored through non-photochemical quenching of chlorophyll fluorescence. When the light intensity is lowered, non-photochemical quenching largely disappears on a time scale ranging from tens of seconds to many minutes. With the use of picosecond fluorescence spectroscopy, we demonstrate that one of the underlying mechanisms is only functional when the reaction centre of photosystem II is closed, that is when electron transfer is blocked and the risk of photodamage is high. This is accompanied by the appearance of a long-wavelength fluorescence band. As soon as the reaction centre reopens, this quenching, together with the long-wavelength fluorescence, disappears instantaneously. This allows plants to maintain a high level of photosynthetic efficiency even in dangerous high-light conditions.

Primary Charge Separation in the Photosystem II Reaction Center Revealed by a Global Analysis of the Two-dimensional Electronic Spectra.

The transfer of electronic charge in the reaction center of Photosystem II is one of the key building blocks of the conversion of sunlight energy into chemical energy within the cascade of the photosynthetic reactions. Since the charge transfer dynamics is mixed with the energy transfer dynamics, an effective tool for the direct resolution of charge separation in the reaction center is still missing. Here, we use experimental two-dimensional optical photon echo spectroscopy in combination with the theoretical calculation to resolve its signature. A global fitting analysis allows us to clearly and directly identify a decay pathway associated to the primary charge separation. In particular, it can be distinguished from regular energy transfer and occurs on a time scale of 1.5 ps under ambient conditions. This technique provides a general tool to identify charge separation signatures from the energy transport in two-dimensional optical spectroscopy.

Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I.

Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem. In higher plants a special pool of LHCII antennae, which can be associated with either PSI or PSII, participates in these state transitions. It is known that one LHCII antenna can associate with the PsaH site of PSI. However, membrane fractions were recently isolated in which multiple LHCII antennae appear to transfer energy to PSI. We have used time-resolved fluorescence-streak camera measurements to investigate the energy transfer rates and efficiency in these membrane fractions. Our data show that energy transfer from LHCII to PSI is relatively slow. Nevertheless, the trapping efficiency in supercomplexes of PSI with ~2.4 LHCIIs attached is 94%. The absorption cross section of PSI can thus be increased with ~65% without having significant loss in quantum efficiency. Comparison of the fluorescence dynamics of PSI-LHCII complexes, isolated in a detergent or located in their native membrane environment, indicates that the environment influences the excitation energy transfer rates in these complexes. This demonstrates the importance of studying membrane protein complexes in their natural environment.

Imaging the Photosystem I/Photosystem II chlorophyll ratio inside the leaf.

Oxygenic photosynthesis is driven by photosystems I (PSI) and II (PSII). In plants the number of chlorophylls of PSI versus PSII is adjusted to the light irradiance spectrum. On a timescale of days, this is regulated at the level of protein concentration. Instead, on a timescale of minutes, it is regulated by the dynamic association of light-harvesting complex II with either PSI or PSII. Thus far very diverse values have been reported for the PSI/PSII chlorophyll ratio, ranging from 0.54 to 1.4. The methods used require the isolation of chloroplasts and are time consuming. We present a fluorescence lifetime imaging approach that quantifies the PSI/PSII Chl ratio of chloroplasts directly in their natural leaf environment. In wild type Arabidopsis thaliana plants, grown under white light, the PSI/PSII chlorophyll ratio appeared to be 0.99±0.09 at the adaxial side and 0.83±0.05 at the abaxial side of the leaf. When these plants were acclimated to far red light for several days the PSI/PSII chlorophyll ratio decreased by more than a factor of 3 to compensate for the ineffective far red light absorption of PSII. This shows how plants optimize their light-harvesting capacity to the specific light conditions they encounter. Zooming in on single chloroplasts inside the leaf allowed to study the grana/stroma membrane network and their PSI/PSII chlorophyll ratios. The developed method will be useful to study dynamic processes in chloroplasts in intact leaves which involve changes in the grana and the stroma membranes such as state transitions.

Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes.