Optimizing photosynthetic light-harvesting under stars: simple and general antenna models.

In the next 10-20 years, several observatories will aim to detect the signatures of oxygenic photosynthesis on exoplanets, though targets must be carefully selected. Most known potentially habitable exo-planets orbit cool M-dwarf stars, which have limited emission in the photosynthetically active region of the spectrum (PAR, 400 < λ < 700 nm) used by Earth's oxygenic photoautotrophs. Still, recent experiments have shown that model cyanobacteria, algae, and non-vascular plants grow comfortably under simulated M-dwarf light, though vascular plants struggle. Here, we hypothesize that this is partly due to the different ways they harvest light, reflecting some general rule that determines how photosynthetic antenna structures may evolve under different stars. We construct a simple thermodynamic model of an oxygenic antenna-reaction centre supercomplex and determine the optimum structure, size and absorption spectrum under light from several star types. For the hotter G (e.g. the Sun) and K-stars, a small modular antenna is optimal and qualitatively resembles the PSII-LHCII supercomplex of higher plants. For the cooler M-dwarfs, a very large antenna with a steep 'energy funnel' is required, resembling the cyanobacterial phycobilisome. For the coolest M-dwarfs an upper limit is reached, where increasing antenna size further is subject to steep diminishing returns in photosynthetic output. We conclude that G- and K-stars could support a range of niches for oxygenic photo-autotrophs, including high-light adapted canopy vegetation that may generate detectable bio-signatures. M-dwarfs may only be able to support low light-adapted organisms that have to invest considerable resources in maintaining a large antenna. This may negatively impact global coverage and therefore detectability.

Expanding the scope of problem-based-learning at Hackensack Meridian School of Medicine; integrating domain-general skills with domain-specific content.

The Master Adaptive Learner is a model used to develop students to become self-regulated and adaptable lifelong learners to practice medicine in a complex and ever-changing environment. The Hackensack Meridian School of Medicine (HMSOM) proposes a new course, Patient Presentation Problem-Based Learning Curriculum (PPPC), a dynamic and integrated course that goes beyond the scope of traditional Problem-Based-Learning (PBL). PPPC allows students to build domain-general skills in tandem with domain-specific content learned during a pre-clerkship curriculum. An integrated case provides weekly scaffolding, such that the course takes place throughout the week and is not isolated from the rest of the curriculum. Students receive iterative feedback and structured assignments which allows development of self-directed learning skills along with integration and consolidation of weekly curricular content. A layered analysis approach was used to outline the philosophies, principles and techniques that link to our course objectives. Techniques used could easily be translated to other pre-clerkship curriculum to promote development of self-directed learning and clinical reasoning skills, as well as promote more meaningful learning of basic, clinical, and health system science content.

Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions.

An intriguing molecular architecture called the "semi-crystalline photosystem II (PSII) array" has been observed in the thylakoid membranes in vascular plants. It is an array of PSII-light-harvesting complex II (LHCII) supercomplexes that only appears in low light, but its functional role has not been clarified. Here, we identified PSII-LHCII supercomplexes in their monomeric and multimeric forms in low light-acclimated spinach leaves and prepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves were acclimated to high light, only the monomeric forms were present, suggesting that the multimeric forms represent a structural adaptation to low light and that disaggregation of the PSII-LHCII supercomplex represents an adaptation to high light. Single-particle EM revealed that the multimeric PSII-LHCII supercomplexes are composed of two ("megacomplex") or three ("arraycomplex") units of PSII-LHCII supercomplexes, which likely constitute a fraction of the semi-crystalline PSII array. Further characterization with fluorescence analysis revealed that multimeric forms have a higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings suggest that the configurational conversion of PSII-LHCII supercomplexes may serve as a structural basis for acclimation of plants to environmental light.

Ultrafast energy transfer between lipid-linked chromophores and plant light-harvesting complex II.

Light-Harvesting Complex II (LHCII) is a membrane protein found in plant chloroplasts that has the crucial role of absorbing solar energy and subsequently performing excitation energy transfer to the reaction centre subunits of Photosystem II. LHCII provides strong absorption of blue and red light, however, it has minimal absorption in the green spectral region where solar irradiance is maximal. In a recent proof-of-principle study, we enhanced the absorption in this spectral range by developing a biohybrid system where LHCII proteins together with lipid-linked Texas Red (TR) chromophores were assembled into lipid membrane vesicles. The utility of these systems was limited by significant LHCII quenching due to protein-protein interactions and heterogeneous lipid structures. Here, we organise TR and LHCII into a lipid nanodisc, which provides a homogeneous, well-controlled platform to study the interactions between TR molecules and single LHCII complexes. Fluorescence spectroscopy determined that TR-to-LHCII energy transfer has an efficiency of at least 60%, resulting in a 262% enhancement of LHCII fluorescence in the 525-625 nm range, two-fold greater than in the previous system. Ultrafast transient absorption spectroscopy revealed two time constants of 3.7 and 128 ps for TR-to-LHCII energy transfer. Structural modelling and theoretical calculations indicate that these timescales correspond to TR-lipids that are loosely- or tightly-associated with the protein, respectively, with estimated TR-to-LHCII separations of ∼3.5 nm and ∼1 nm. Overall, we demonstrate that a nanodisc-based biohybrid system provides an idealised platform to explore the photophysical interactions between extrinsic chromophores and membrane proteins with potential applications in understanding more complex natural or artificial photosynthetic systems.

Excitation quenching in chlorophyll-carotenoid antenna systems: 'coherent' or 'incoherent'.

Plants possess an essential ability to rapidly down-regulate light-harvesting in response to high light. This photoprotective process involves the formation of energy-quenching interactions between the chlorophyll and carotenoid pigments within the antenna of Photosystem II (PSII). The nature of these interactions is currently debated, with, among others, 'incoherent' or 'coherent' quenching models (or a combination of the two) suggested by a range of time-resolved spectroscopic measurements. In 'incoherent quenching', energy is transferred from a chlorophyll to a carotenoid and is dissipated due to the intrinsically short excitation lifetime of the latter. 'Coherent quenching' would arise from the quantum mechanical mixing of chlorophyll and carotenoid excited state properties, leading to a reduction in chlorophyll excitation lifetime. The key parameters are the energy gap, [Formula: see text] and the resonance coupling, J, between the two excited states. Coherent quenching will be the dominant process when [Formula: see text] i.e., when the two molecules are resonant, while the quenching will be largely incoherent when [Formula: see text] One would expect quenching to be energetically unfavorable for [Formula: see text] The actual dynamics of quenching lie somewhere between these limiting regimes and have non-trivial dependencies of both J and [Formula: see text] Using the Hierarchical Equation of Motion (HEOM) formalism we present a detailed theoretical examination of these excitation dynamics and their dependence on slow variations in J and [Formula: see text] We first consider an isolated chlorophyll-carotenoid dimer before embedding it within a PSII antenna sub-unit (LHCII). We show that neither energy transfer, nor the mixing of excited state lifetimes represent unique or necessary pathways for quenching and in fact discussing them as distinct quenching mechanisms is misleading. However, we do show that quenching cannot be switched 'on' and 'off' by fine tuning of [Formula: see text] around the resonance point, [Formula: see text] Due to the large reorganization energy of the carotenoid excited state, we find that the presence (or absence) of coherent interactions have almost no impact of the dynamics of quenching. Counter-intuitively significant quenching is present even when the carotenoid excited state lies above that of the chlorophyll. We also show that, above a rather small threshold value of [Formula: see text]quenching becomes less and less sensitive to J (since in the window [Formula: see text] the overall lifetime is independent of it). The requirement for quenching appear to be only that [Formula: see text] Although the coherent/incoherent character of the quenching can vary, the overall kinetics are likely robust with respect to fluctuations in J and [Formula: see text] This may be the basis for previous observations of NPQ with both coherent and incoherent features.

How carotenoid distortions may determine optical properties: lessons from the Orange Carotenoid Protein.

Carotenoids in photosynthetic proteins carry out the dual function of harvesting light and defending against photo-damage by quenching excess energy. The latter involves the low-lying, dark, excited state labelled S1. Here "dark" means optically-forbidden, a property that is often attributed to molecular symmetry, which leads to speculation that its optical properties may be strongly-perturbed by structural distortions. This has been both explicitly and implicitly proposed as an important feature of photo-protective energy quenching. Here we present a theoretical analysis of the relationship between structural distortions and S1 optical properties. We outline how S1 is dark not because of overall geometric symmetry but because of a topological symmetry related to bond length alternation in the conjugated backbone. Taking the carotenoid echinenone as an example and using a combination of molecular dynamics, quantum chemistry, and the theory of spectral lineshapes, we show that distortions that break this symmetry are extremely stiff. They are therefore absent in solution and only marginally present in even a very highly-distorted protein binding pocket such as in the Orange Carotenoid Protein (OCP). S1 remains resolutely optically-forbidden despite any breaking of bulk molecular symmetry by the protein environment. However, rotations of partially conjugated end-rings can result in fine tuning of the S1 transition density which may exert some influence on interactions with neighbouring chromophores.

Research questions to facilitate the future development of European long-term ecosystem research infrastructures: A horizon scanning exercise.

Distributed environmental research infrastructures are important to support assessments of the effects of global change on landscapes, ecosystems and society. These infrastructures need to provide continuity to address long-term change, yet be flexible enough to respond to rapid societal and technological developments that modify research priorities. We used a horizon scanning exercise to identify and prioritize emerging research questions for the future development of ecosystem and socio-ecological research infrastructures in Europe. Twenty research questions covered topics related to (i) ecosystem structures and processes, (ii) the impacts of anthropogenic drivers on ecosystems, (iii) ecosystem services and socio-ecological systems and (iv), methods and research infrastructures. Several key priorities for the development of research infrastructures emerged. Addressing complex environmental issues requires the adoption of a whole-system approach, achieved through integration of biotic, abiotic and socio-economic measurements. Interoperability among different research infrastructures needs to be improved by developing standard measurements, harmonizing methods, and establishing capacities and tools for data integration, processing, storage and analysis. Future research infrastructures should support a range of methodological approaches including observation, experiments and modelling. They should also have flexibility to respond to new requirements, for example by adjusting the spatio-temporal design of measurements. When new methods are introduced, compatibility with important long-term data series must be ensured. Finally, indicators, tools, and transdisciplinary approaches to identify, quantify and value ecosystem services across spatial scales and domains need to be advanced.

A possible molecular basis for photoprotection in the minor antenna proteins of plants.

The bioenergetics of light-harvesting by photosynthetic antenna proteins in higher plants is well understood. However, investigation into the regulatory non-photochemical quenching (NPQ) mechanism, which dissipates excess energy in high light, has led to several conflicting models. It is generally accepted that the major photosystem II antenna protein, LHCII, is the site of NPQ, although the minor antenna complexes (CP24/26/29) are also proposed as alternative/additional NPQ sites. LHCII crystals were shown to exhibit the short excitation lifetime and several spectral signatures of the quenched state. Subsequent structure-based models showed that this quenching could be explained by slow energy trapping by the carotenoids, in line with one of the proposed models. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we show that the crystal structure of CP29 corresponds to a strongly quenched conformation. Using a structure-based theoretical model we show that this quenching may be explained by the same slow, carotenoid-mediated quenching mechanism present in LHCII crystals.

The carotenoid pathway: what is important for excitation quenching in plant antenna complexes?

Plant light-harvesting is regulated by the Non-Photochemical Quenching (NPQ) mechanism involving the reversible formation of excitation quenching sites in the Photosystem II (PSII) antenna in response to high light. While the major antenna complex, LHCII, is known to be a site of NPQ, the precise mechanism of excitation quenching is not clearly understood. A preliminary model of the quenched crystal structure of LHCII implied that quenching arises from slow energy capture by Car pigments. It predicted a thoroughly quenched system but offered little insight into the defining aspects of this quenching. In this work, we present a thorough theoretical investigation of this quenching, addressing the factors defining the quenching pathway and possible mechanism for its (de)activation. We show that quenching in LHCII crystals is the result of slow energy transfer from chlorophyll to the centrally-bound lutein Cars, predominantly the Lut620 associated with the chlorophyll 'terminal emitter', one of the proposed in vivo pathways. We show that this quenching is rather independent of the particular species of Car and excitation 'site' energy. The defining parameter is the resonant coupling between the pigment co-factors. Lastly, we show that these interactions must be severely suppressed for a light-harvesting state to be recovered.

New Paths to Professional Nursing: Using Encouragement to Prepare a Minority Workforce to Enter the Nursing Profession.

The current professional nursing workforce in the United States is predominantly White and female, even though minorities compose 33% of the national population (Travers, Smaldone, & Cohn, 2015). Minority patients are more effectively cared for when their particular cultural milieu is taken into consideration as part of their health care plan (Sullivan, 2004). According to the Agency for Healthcare Research and Quality (AHRQ), health care quality remains suboptimal for diverse populations in the United States because some individuals do not receive quality care or do not believe their values are honored or respected (AHRQ, 2016). Minority professional nurses are necessary to address the racial and ethnic disparities in health care. Robert Wood Johnson Barnabas Health (RWJBH), in collaboration with Rutgers University School of Nursing (RUSON), implemented New Paths to Professional Nursing (NPPN) to increase the number of minority professional nurses in practice at RWJBH. The program provided financial resources as well as infrastructural, group, and personal support for RWJBH minority employees who desired to complete prerequisites to enter RUSON. The academic success of the employees who participated in NPPN was attributed to a unique combination of financial assistance and support and encouragement. The purpose of this article is to describe in detail the development of the program and the effective encouragement strategies that have led to success for NPPN minority student/employees. This article examines, defines, and illustrates particular types of effective encouragement and suggests that this encouragement was the key to success for the NPPN minority students.

An 'all pigment' model of excitation quenching in LHCII.

The rapid, photoprotective down-regulation of plant light-harvesting in bright light proceeds via the non-photochemical quenching of chlorophyll excitation energy in the major photosystem II light-harvesting complex LHCII. However, there is currently no consensus regarding the precise mechanism by which excess energy is quenched. Current X-ray structures of this complex correspond to a dissipative conformation and therefore correct microscopic theoretical modelling should capture this property. Despite their accuracy in explaining the steady state spectroscopy of this complex, chlorophyll-only models (those that neglect the energetic role of carotenoids) do not explain the observed fluorescence quenching. To address this gap, we have used a combination of the semi-empirical MNDO-CAS-CI and the Transition Density Cube method to model all chlorophyll-carotenoid energy transfer pathways in the highly quenched LHCII X-ray structure. Our simulations reveal that the inclusion of carotenoids in this microscopic model results in profound excitation quenching, reducing the predicted excitation lifetime of the complex from 4 ns (chlorophyll-only) to 67 ps. The model indicates that energy dissipation proceeds via slow excitation transfer (>20 ps) from chlorophyll to the forbidden S1 excited state of the centrally bound lutein molecules followed by the rapid (∼10 ps) radiationless decay to the ground state, with the latter being assumed from experimental measurements of carotenoid excited state lifetimes. Violaxanthin and neoxanthin do not contribute to this quenching. This work presents the first all-pigment microscopic model of LHCII and the first attempt to capture the dissipative character of the known structure.

Photoprotective energy dissipation involves the reorganization of photosystem II light-harvesting complexes in the grana membranes of spinach chloroplasts.

Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

Modeling the NMR signatures associated with the functional conformational switch in the major light-harvesting antenna of photosystem II in higher plants.

The major photosystem II antenna complex, LHCII, possesses an intrinsic conformational switch linked to the formation of a photoprotective, excitation-quenching state. Recent solid state NMR experiments revealed that aggregation-induced quenching in (13)C-enriched LHCII from C. reinhardtii is associated with changes to the chemical shifts of three specific (13)C atoms in the Chla conjugated macrocycle. We performed DFT-based NMR calculations on the strongly-quenched crystal structure of LHCII (taken from spinach). We demonstrate that specific Chla-xanthophyll interactions in the quenched structure lead to changes in the Chla(13)C chemical shifts that are qualitatively similar to those observed by solid state NMR. We propose that these NMR changes are due to modulations in Chla-xanthophyll associations that occur due to a quenching-associated functional conformation change in the lutein and neoxanthin domains of LHCII. The combination of solid-state NMR and theoretical modeling is therefore a powerful tool for assessing functional conformational switching in the photosystem II antenna.

Unravelling the fluorescence kinetics of light-harvesting proteins with simulated measurements.

The plant light-harvesting pigment-protein complex LHCII is the major antenna sub-unit of PSII and is generally (though not universally) accepted to play a role in photoprotective energy dissipation under high light conditions, a process known Non-Photochemical Quenching (NPQ). The underlying mechanisms of energy trapping and dissipation within LHCII are still debated. Various models have been proposed for the underlying molecular detail of NPQ, but they are often based on different interpretations of very similar transient absorption measurements of isolated complexes. Here we present a simulated measurement of the fluorescence decay kinetics of quenched LHCII aggregates to determine whether this relatively simple measurement can discriminate between different potential NPQ mechanisms. We simulate not just the underlying physics (excitation, energy migration, quenching and singlet-singlet annihilation) but also the signal detection and typical experimental data analysis. Comparing this to a selection of published fluorescence decay kinetics we find that: (1) Different proposed quenching mechanisms produce noticeably different fluorescence kinetics even at low (annihilation free) excitation density, though the degree of difference is dependent on pulse width. (2) Measured decay kinetics are consistent with most LHCII trimers becoming relatively slow excitation quenchers. A small sub-population of very fast quenchers produces kinetics which do not resemble any observed measurement. (3) It is necessary to consider at least two distinct quenching mechanisms in order to accurately reproduce experimental kinetics, supporting the idea that NPQ is not a simple binary switch.

The photoprotective molecular switch in the photosystem II antenna.

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II.

Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis.

We characterized a set of Arabidopsis mutants deficient in specific light-harvesting proteins, using freeze-fracture electron microscopy to probe the organization of complexes in the membrane and confocal fluorescence recovery after photobleaching to probe the dynamics of thylakoid membranes within intact chloroplasts. The same methods were used to characterize mutants lacking or over-expressing PsbS, a protein related to light-harvesting complexes that appears to play a role in regulation of photosynthetic light harvesting. We found that changes in the complement of light-harvesting complexes and PsbS have striking effects on the photosystem II macrostructure, and that these effects correlate with changes in the mobility of chlorophyll proteins within the thylakoid membrane. The mobility of chlorophyll proteins was found to correlate with the extent of photoprotective non-photochemical quenching, consistent with the idea that non-photochemical quenching involves extensive re-organization of complexes in the membrane. We suggest that a key feature of the physiological function of PsbS is to decrease the formation of ordered semi-crystalline arrays of photosystem II in the low-light state. Thus the presence of PsbS leads to an increase in the fluidity of the membrane, accelerating the re-organization of the photosystem II macrostructure that is necessary for induction of non-photochemical quenching.

Switching light harvesting complex II into photoprotective state involves the lumen-facing apoprotein loop.

In higher plants, high light conditions trigger the activation of non-photochemical quenching (NPQ), a process of photoprotective light energy dissipation, via acidification of the chloroplast lumen. Spectral changes occurring in the neoxanthin domain of the major light harvesting antenna complex (LHCII) have previously provided indirect evidence of a protein conformational switch during NPQ. We report here of two recombinant LHCII complexes mutated at the level of lumenal loop with altered quenching capacity with respect to the control. Replacement of the acidic lumenal-facing residue aspartate 111 (D111) with neutral valine (V111) yielded a recombinant complex with increased quenching capacity under low pH, due to a shift of the pK by 1 pH unit. The increase in total quenching was consistent with 40% reduction in the relative chlorophyll fluorescence lifetime and was accompanied by a lower energy emitting state of the mutant, as demonstrated by 77 K fluorescence spectroscopy. On the other hand, replacement of acidic glutamate 94 (E94) with glycine (G94) resulted in reduction of the fluorescence quenching yield attained at low pH. These results show for the first time that a subtle change in the LHCII apoprotein structure at the level of the lumenal loop induced by single aminoacid mutagenesis can affect protein sensitivity to pH leading to the establishment of NPQ. This work opens a potential avenue for manipulation of light harvesting efficiency in the natural antenna pigment-protein complexes that can be used for the creation of hybrid light energy conversion systems in future.

Introducing Life-long Learning Skills in a Patient Presentation Problem-Based Curriculum: The Case for Librarian Integration.

Medical students must learn how to find, critically appraise, and apply high-quality information to support their clinical decisions. To reinforce these skills, the Hackensack Meridian School of Medicine embedded medical librarians into a longitudinal case-based, problem-based learning curriculum, where they provide individualized feedback on student's skills in this area.

Origin of absorption changes associated with photoprotective energy dissipation in the absence of zeaxanthin.

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.