RESUMEN
The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Complejos de Proteína Captadores de Luz , Fotosíntesis , Complejo de Proteína del Fotosistema II , Tilacoides , Tilacoides/metabolismo , Tilacoides/química , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/química , Galactolípidos/metabolismo , Galactolípidos/química , LuzRESUMEN
Plant tolerance to high light and oxidative stress is increased by overexpression of the photosynthetic enzyme Ferredoxin:NADP(H) reductase (FNR), but the specific mechanism of FNR-mediated protection remains enigmatic. It has also been reported that the localization of this enzyme within the chloroplast is related to its role in stress tolerance. Here, we dissected the impact of FNR content and location on photoinactivation of photosystem I (PSI) and photosystem II (PSII) during high light stress of Arabidopsis (Arabidopsis thaliana). The reaction center of PSII is efficiently turned over during light stress, while damage to PSI takes much longer to repair. Our results indicate a PSI sepcific effect, where efficient oxidation of the PSI primary donor (P700) upon transition from darkness to light, depends on FNR recruitment to the thylakoid membrane tether proteins: thylakoid rhodanase-like protein (TROL) and translocon at the inner envelope of chloroplasts 62 (Tic62). When these interactions were disrupted, PSI photoinactivation occurred. In contrast, there was a moderate delay in the onset of PSII damage. Based on measurements of ΔpH formation and cyclic electron flow, we propose that FNR location influences the speed at which photosynthetic control is induced, resulting in specific impact on PSI damage. Membrane tethering of FNR therefore plays a role in alleviating high light stress, by regulating electron distribution during short-term responses to light.
Asunto(s)
Adaptación Ocular/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ferredoxina-NADP Reductasa/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Adaptación Ocular/genética , Cloroplastos/genética , Ferredoxina-NADP Reductasa/genética , Variación Genética , Genotipo , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema II/genéticaRESUMEN
Light harvesting is regulated by a process triggered by the acidification of the thylakoid lumen, known as nonphotochemical "energy-dependent quenching" (qE). In diatoms, qE is controlled by the light-harvesting complex (LHC) protein LHCX1, while the LHC stress-related (LHCSR) and photosystem II subunit S proteins are essential for green algae and plants, respectively. Here, we report a biochemical and molecular characterization of LHCX1 to investigate its role in qE. We found that, when grown under intermittent light, Phaeodactylum tricornutum forms very large qE, due to LHCX1 constitutive upregulation. This "super qE" is abolished in LHCX1 knockout mutants. Biochemical and spectroscopic analyses of LHCX1 reveal that this protein might differ in the character of binding pigments relative to the major pool of light-harvesting antenna proteins. The possibility of transient pigment binding or not binding pigments at all is discussed. Targeted mutagenesis of putative protonatable residues (D95 and E205) in transgenic P. tricornutum lines does not alter qE capacity, showing that they are not involved in sensing lumen pH, differently from residues conserved in LHCSR3. Our results suggest functional divergence between LHCX1 and LHCSR3 in qE modulation. We propose that LHCX1 evolved independently to facilitate dynamic tracking of light fluctuations in turbulent waters. The evolution of LHCX(-like) proteins in organisms with secondary red plastids, such as diatoms, might have conferred a selective advantage in the control of dynamic photoprotection, ultimately resulting in their ecological success.
Asunto(s)
Adaptación Fisiológica/genética , Diatomeas/genética , Diatomeas/metabolismo , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de PlantasRESUMEN
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.
Asunto(s)
Complejos de Proteína Captadores de Luz , Fotosíntesis , Adaptación Fisiológica , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis/fisiología , Plantas/metabolismo , Tilacoides/metabolismoRESUMEN
Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Clorofila A/metabolismo , Fotosíntesis/genética , Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismoRESUMEN
The photosystem II reaction centre (RCII) protein subunit D1 is the main target of light-induced damage in the thylakoid membrane. As such, it is constantly replaced with newly synthesised proteins, in a process dubbed the 'D1 repair cycle'. The mechanism of relief of excitation energy pressure on RCII, non-photochemical quenching (NPQ), is activated to prevent damage. The contribution of the D1 repair cycle and NPQ in preserving the photochemical efficiency of RCII is currently unclear. In this work, we seek to (1) quantify the relative long-term effectiveness of photoprotection offered by NPQ and the D1 repair cycle, and (2) determine the fraction of sustained decrease in RCII activity that is due to long-term protective processes. We found that while under short-term, sunfleck-mimicking illumination, NPQ is substantially more effective in preserving RCII activity than the D1 repair cycle (Plant. Cell Environ.41, 1098-1112, 2018). Under prolonged constant illumination, its contribution is less pronounced, accounting only for up to 30% of RCII protection, while D1 repair assumes a predominant role. Exposure to a wide range of light intensities yields comparable results, highlighting the crucial role of a constant and rapid D1 turnover for the maintenance of RCII efficiency. The interplay between NPQ and D1 repair cycle is crucial to grant complete phototolerance to plants under low and moderate light intensities, and limit damage to photosystem II under high light. Additionally, we disentangled and quantified the contribution of a slowly reversible NPQ component that does not impair RCII activity, and is therefore protective.
Asunto(s)
Complejo de Proteína del Fotosistema II , Tilacoides , Luz , Células Vegetales , Subunidades de ProteínaRESUMEN
Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H+ /K+ antiporter KEA3 in the model diatom Phaeodactylum tricornutum. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca2+ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.
Asunto(s)
Diatomeas , Aclimatación , Diatomeas/metabolismo , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis , ProtonesRESUMEN
The proton motive force (pmf) across the thylakoid membrane couples photosynthetic electron transport and ATP synthesis. In recent years, the electrochromic carotenoid and chlorophyll absorption band shift (ECS), peaking â¼515 nm, has become a widely used probe to measure pmf in leaves. However, the use of this technique to calculate the parsing of the pmf between the proton gradient (ΔpH) and electric potential (Δψ) components remains controversial. Interpretation of the ECS signal is complicated by overlapping absorption changes associated with violaxanthin de-epoxidation to zeaxanthin (ΔA505) and energy-dependent nonphotochemical quenching (qE; ΔA535). In this study, we used Arabidopsis (Arabidopsis thaliana) plants with altered xanthophyll cycle activity and photosystem II subunit S (PsbS) content to disentangle these overlapping contributions. In plants where overlap among ΔA505, ΔA535, and ECS is diminished, such as npq4 (lacking ΔA535) and npq1npq4 (also lacking ΔA505), the parsing method implies the Δψ contribution is virtually absent and pmf is solely composed of ΔpH. Conversely, in plants where ΔA535 and ECS overlap is enhanced, such as L17 (a PsbS overexpressor) and npq1 (where ΔA535 is blue-shifted to 525 nm) the parsing method implies a dominant contribution of Δψ to the total pmf. These results demonstrate the vast majority of the pmf attributed by the ECS parsing method to Δψ is caused by ΔA505 and ΔA535 overlap, confirming pmf is dominated by ΔpH following the first 60 s of continuous illumination under both low and high light conditions. Further implications of these findings for the regulation of photosynthesis are discussed.
Asunto(s)
Arabidopsis/fisiología , Luz , Fotosíntesis , Fuerza Protón-Motriz , Concentración de Iones de HidrógenoRESUMEN
Photosystem II (PSII) uses light energy to split water into protons, electrons, and oxygen, ultimately sustaining heterotrophic life on Earth. The major light harvesting complex in plants (LHCII) is packed with chlorophylls and carotenoids and is the main supplier of excitation energy to PSII reaction centers. The protein scaffold acts as a programmed solvent for the pigments in LHCII, tuning their orientations while at the same time impeding concentration quenching to ensure efficient storage of excitation energy by chlorophylls. However, under stress, the very fuel of PSII, solar photons, can damage its delicate inner components and hamper photosynthesis. In a crucial regulatory strategy in plants, LHCII evolved a flexible design that allows it to switch between light-harvesting and dissipative conformations, thereby safely releasing the excess energy that is absorbed into heat. Several mechanisms have been proposed to explain chlorophyll de-excitation pathways in LHCII, such as chlorophyll-chlorophyll charge transfer states, resonance energy transfer from chlorophylls to a carotenoid S1 state, and chlorophyll-carotenoid reductive energy transfer. This Perspective critically assesses the listed proposals, addressing both the physical mechanism of quenching and the nature of the quenching pigment. These hypotheses are then discussed in the context of state-of-the-art biochemical, physiological, and genetic knowledge to scrutinize their likeliness to occur in the native thylakoid membranes.
RESUMEN
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.
Asunto(s)
Chlamydomonas reinhardtii/química , Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/química , Hojas de la Planta/química , Aclimatación , Chlamydomonas reinhardtii/fisiología , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/ultraestructura , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/ultraestructura , Hojas de la Planta/fisiología , Conformación Proteica , Multimerización de Proteína , Tilacoides/química , Tilacoides/metabolismoRESUMEN
Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.
Asunto(s)
Arabidopsis/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Tilacoides/metabolismo , Arabidopsis/efectos de los fármacos , Cinética , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Lincomicina/farmacología , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/metabolismo , Multimerización de Proteína , Espectrometría de Fluorescencia , Ultracentrifugación , Xantófilas/química , Xantófilas/metabolismo , Zeaxantinas/química , Zeaxantinas/metabolismoRESUMEN
Photoprotection refers to a set of well defined plant processes that help to prevent the deleterious effects of high and excess light on plant cells, especially within the chloroplast. Molecular components of chloroplast photoprotection are closely aligned with those of photosynthesis and together they influence productivity. Proof of principle now exists that major photoprotective processes such as non-photochemical quenching (NPQ) directly determine whole canopy photosynthesis, biomass and yield via prevention of photoinhibition and a momentary downregulation of photosynthetic quantum yield. However, this phenomenon has neither been quantified nor well characterized across different environments. Here we address this problem by assessing the existing literature with a different approach to that taken previously, beginning with our understanding of the molecular mechanism of NPQ and its regulation within dynamic environments. We then move to the leaf and the plant level, building an understanding of the circumstances (when and where) NPQ limits photosynthesis and linking to our understanding of how this might take place on a molecular and metabolic level. We argue that such approaches are needed to fine tune the relevant features necessary for improving dynamic NPQ in important crop species.
Asunto(s)
Hojas de la Planta/metabolismo , Fenómenos Fisiológicos de las Plantas , Dióxido de Carbono/metabolismo , Procesos Fotoquímicos , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Non-photochemical chlorophyll fluorescence quenching (NPQ) remains one of the most studied topics of the 21st century in photosynthesis research. Over the past 30 years, profound knowledge has been obtained on the molecular mechanism of NPQ in higher plants. First, the largely overlooked significance of NPQ in protecting the reaction center of photosystem II (RCII) against damage, and the ways to assess its effectiveness are highlighted. Then, the key in vivo signals that can monitor the life of the major NPQ component, qE, are presented. Finally, recent knowledge on the site of qE and the possible molecular events that transmit ΔpH into the conformational change in the major LHCII [the major trimeric light harvesting complex of photosystem II (PSII)] antenna complex are discussed. Recently, number of reports on Arabidopsis mutants lacking various antenna components of PSII confirmed that the in vivo site of qE rests within the major trimeric LHCII complex. Experiments on biochemistry, spectroscopy, microscopy and molecular modeling suggest an interplay between thylakoid membrane geometry and the dynamics of LHCII, the PsbS (PSII subunit S) protein and thylakoid lipids. The molecular basis for the qE-related conformational change in the thylakoid membrane, including the possible onset of a hydrophobic mismatch between LHCII and lipids, potentiated by PsbS protein, begins to unfold.
Asunto(s)
Procesos Fotoquímicos , Plantas/metabolismo , Luz/efectos adversos , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Fenómenos Fisiológicos de las PlantasRESUMEN
Under blue light, plant chloroplasts relocate to different areas of the cell. The photoreceptor phototropin2 (phot2) mediates the chloroplast movement mechanism under excess blue light alongside the chloroplast unusual positioning1 (chup1) protein. Recently, it has been proposed that leaf transmittance changes associated with chloroplast relocation affect measurements of nonphotochemical quenching (NPQ), resulting in kinetic differences due to these movements (termed "qM"). We evaluated these claims using Arabidopsis (Arabidopsis thaliana) knock-out mutants lacking either phot2 or chup1 and analyzed the kinetics of both the onset and recovery of NPQ under equivalent intensities of both red and blue light. We also evaluated the photoprotective ability of chloroplast movements both during the early onset of photoinhibition and under sustained excess light. We monitored photoinhibition using the chlorophyll fluorescence parameter of photochemical quenching in the dark, which measures the redox state of QA within PSII without any of the complications of traditional F v /F m measurements. While there were noticeable differences between the responses under red and blue light, the chloroplast movement mechanism had no effect on the rate or amplitude of NPQ onset or recovery. Therefore, we were unable to replicate the "qM" component and its corresponding influence on the kinetics of NPQ in Arabidopsis grown under "shade" conditions. Furthermore, chloroplast relocation had no effect on the high-light tolerance of these plants. These data cast doubt upon the existence of a chloroplast movement-dependent component of NPQ Therefore, the influence of chloroplast movements on photoprotection should be thoroughly reevaluated.
Asunto(s)
Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Luz , Procesos Fotoquímicos/efectos de la radiación , Cinética , Movimiento , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
Photosystems possess distinct fluorescence emissions at low (77K) temperature. PSI emits in the long-wavelength region at ~710-740 nm. In diatoms, a successful clade of marine primary producers, the contribution of PSI-associated emission (710-717 nm) has been shown to be relatively small. However, in the pennate diatom Phaeodactylum tricornutum, the source of the long-wavelength emission at ~710 nm (F710) remains controversial. Here, we addressed the origin and modulation of F710 fluorescence in this alga grown under continuous and intermittent light. The latter condition led to a strong enhancement in F710. Biochemical and spectral properties of the photosynthetic complexes isolated from thylakoid membranes were investigated for both culture conditions. F710 emission appeared to be associated with PSI regardless of light acclimation. To further assess whether PSII could also contribute to this emission, we decreased the concentration of PSII reaction centres and core antenna by growing cells with lincomycin, a chloroplast protein synthesis inhibitor. The treatment did not diminish F710 fluorescence. Our data suggest that F710 emission originates from PSI under the conditions tested and is enhanced in intermittent light-grown cells due to increased energy flow from the FCP antenna to PSI.
Asunto(s)
Diatomeas , Complejo de Proteína del Fotosistema I , Clorofila , Cloroplastos/metabolismo , Diatomeas/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismoRESUMEN
Photosynthesis in plants starts with the capture of photons by light-harvesting complexes (LHCs). Structural biology and spectroscopy approaches have led to a map of the architecture and energy transfer pathways between LHC pigments. Still, controversies remain regarding the role of specific carotenoids in light-harvesting and photoprotection, obligating the need for high-resolution techniques capable of identifying excited-state signatures and molecular identities of the various pigments in photosynthetic systems. Here we demonstrate the successful application of femtosecond stimulated Raman spectroscopy (FSRS) to a multichromophoric biological complex, trimers of LHCII. We demonstrate the application of global and target analysis (GTA) to FSRS data and utilize it to quantify excitation migration in LHCII trimers. This powerful combination of techniques allows us to obtain valuable insights into structural, electronic, and dynamic information from the carotenoids of LHCII trimers. We report spectral and dynamical information on ground- and excited-state vibrational modes of the different pigments, resolving the vibrational relaxation of the carotenoids and the pathways of energy transfer to chlorophylls. The lifetimes and spectral characteristics obtained for the S1 state confirm that lutein 2 has a distorted conformation in LHCII and that the lutein 2 S1 state does not transfer to chlorophylls, while lutein 1 is the only carotenoid whose S1 state plays a significant energy-harvesting role. No appreciable energy transfer takes place from lutein 1 to lutein 2, contradicting recent proposals regarding the functions of the various carotenoids (Son et al. Chem. 2019, 5 (3), 575-584). Also, our results demonstrate that FSRS can be used in combination with GTA to simultaneously study the electronic and vibrational landscapes in LHCs and pave the way for in-depth studies of photoprotective conformations in photosynthetic systems.
RESUMEN
Plants are subject to dramatic fluctuations in the intensity of sunlight throughout the day. When the photosynthetic machinery is exposed to high light, photons are absorbed in excess, potentially leading to oxidative damage of its delicate membrane components. A photoprotective molecular process called non-photochemical quenching (NPQ) is the fastest response carried out in the thylakoid membranes to harmlessly dissipate excess light energy. Despite having been intensely studied, the site and mechanism of this essential regulatory process are still debated. Here, we show that the main NPQ component called energy-dependent quenching (qE) is present in plants with photosynthetic membranes largely enriched in the major trimeric light-harvesting complex (LHC) II, while being deprived of all minor LHCs and most photosystem core proteins. This fast and reversible quenching depends upon thylakoid lumen acidification (ΔpH). Enhancing ΔpH amplifies the extent of the quenching and restores qE in the membranes lacking PSII subunit S protein (PsbS), whereas the carotenoid zeaxanthin modulates the kinetics and amplitude of the quenching. These findings highlight the self-regulatory properties of the photosynthetic light-harvesting membranes in vivo, where the ability to switch reversibly between the harvesting and dissipative states is an intrinsic property of the major LHCII.
Asunto(s)
Arabidopsis , Complejos de Proteína Captadores de Luz , Arabidopsis/metabolismo , Clorofila , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Xantófilas/metabolismo , Zeaxantinas/metabolismoRESUMEN
High light intensities raise photosynthetic and plant growth rates but can cause damage to the photosynthetic machinery. The likelihood and severity of deleterious effects are minimised by a set of photoprotective mechanisms, one key process being the controlled dissipation of energy from chlorophyll within PSII known as non-photochemical quenching (NPQ). Although ubiquitous, the role of NPQ in plant productivity is important because it momentarily reduces the quantum efficiency of photosynthesis. Rice plants overexpressing and deficient in the gene encoding a central regulator of NPQ, the protein PsbS, were used to assess the effect of protective effectiveness of NPQ (pNPQ) at the canopy scale. Using a combination of three-dimensional reconstruction, modelling, chlorophyll fluorescence, and gas exchange, the influence of altered NPQ capacity on the distribution of pNPQ was explored. A higher phototolerance in the lower layers of a canopy was found, regardless of genotype, suggesting a mechanism for increased protection for leaves that experience relatively low light intensities interspersed with brief periods of high light. Relative to wild-type plants, psbS overexpressors have a reduced risk of photoinactivation and early growth advantage, demonstrating that manipulating photoprotective mechanisms can impact both subcellular mechanisms and whole-canopy function.
Asunto(s)
Oryza , Clorofila , Luz , Oryza/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismoRESUMEN
Xanthophylls in light harvesting complexes perform a number of functions ranging from structural support to light-harvesting and photoprotection. In the major light harvesting complex of photosystem II in plants (LHCII), the innermost xanthophyll binding pockets are occupied by lutein molecules. The conservation of these sites within the LHC protein family suggests their importance in LHCII functionality. In the present work, we induced the photoprotective switch in LHCII isolated from the Arabidopsis mutant npq1lut2, where the lutein molecules are exchanged with violaxanthin. Despite the differences in the energetics of the pigments and the impairment of chlorophyll fluorescence quenching in vivo, we show that isolated complexes containing violaxanthin are still able to induce the quenching switch to a similar extent to wild type LHCII monomers. Moreover, the same spectroscopic changes take place, which suggest the involvement of the terminal emitter site (L1) in energy dissipation in both complexes. These results indicate the robust nature of the L1 xanthophyll binding domain in LHCII, where protein structural cues are the major determinant of the function of the bound carotenoid.
Asunto(s)
Complejo de Proteína del Fotosistema II/metabolismo , Xantófilas/metabolismo , Arabidopsis/química , Luteína/química , Luteína/metabolismo , Procesos Fotoquímicos , Complejo de Proteína del Fotosistema II/química , Xantófilas/químicaRESUMEN
Photoinhibition is the light-induced down-regulation of photosynthetic efficiency, the primary target of which is photosystem II (PSII). Currently, there is no clear consensus on the exact mechanism of this process. However, it is clear that inhibition can occur through limitations on both the acceptor- and donor side of PSII. The former mechanism is caused by electron transport limitations at the PSII acceptor side. Whilst, the latter mechanism relies on the disruption of the oxygen-evolving complex. Both of these mechanisms damage the PSII reaction centre (RC). Using a novel chlorophyll fluorescence methodology, RC photoinactivation can be sensitively measured and quantified alongside photoprotection in vivo This is achieved through estimation of the redox state of QA, using the parameter of photochemical quenching in the dark (qPd). This study shows that through the use of PSII donor-side inhibitors, such as UV-B and Cd2+, there is a steeper gradient of photoinactivation in the systems with a weakened donor side, independent of the level of NPQ attained. This is coupled with a concomitant decline in the light tolerance of PSII. The native light tolerance is partially restored upon the use of 1,5-diphenylcarbazide (DPC), a PSII electron donor, allowing for the balance between the inhibitory pathways to be sensitively quantified. Thus, this study confirms that the impact of donor-side inhibition can be detected alongside acceptor-side photoinhibition using the qPd parameter and confirms qPd as a valid, sensitive and unambiguous parameter to sensitively quantify the onset of photoinhibition through both acceptor- or donor-side mechanisms.