RESUMO
Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.
Assuntos
Proteínas de Algas/química , Proteínas de Algas/metabolismo , Fluorescência , Fotossíntese , Cinética , Multimerização Proteica , Transporte Proteico , Espectrometria de FluorescênciaRESUMO
Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.
Assuntos
Proteína do Homeodomínio de Antennapedia/genética , Complexos de Proteínas Captadores de Luz/genética , Processos Fototróficos/genética , Agregados Proteicos/genética , Proteína do Homeodomínio de Antennapedia/química , Clorofila/química , Clorofila/genética , Clorofila/efeitos da radiação , Análise por Conglomerados , Fluorescência , Concentração de Íons de Hidrogênio , Luz/efeitos adversos , Complexos de Proteínas Captadores de Luz/química , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/efeitos da radiação , Espectrometria de Fluorescência , Tilacoides/química , Tilacoides/genética , Tilacoides/efeitos da radiação , Zeaxantinas/genéticaRESUMO
In the present paper, we report an improved method combining sucrose density gradient with ion-exchange chromatography for the isolation of pure chlorophyll a/c antenna proteins from the model cryptophytic alga Rhodomonas salina. Antennas were used for in vitro quenching experiments in the absence of xanthophylls, showing that protein aggregation is a plausible mechanism behind non-photochemical quenching in R. salina. From sucrose gradient, it was also possible to purify a functional photosystem I supercomplex, which was in turn characterized by steady-state and time-resolved fluorescence spectroscopy. R. salina photosystem I showed a remarkably fast photochemical trapping rate, similar to what recently reported for other red clade algae such as Chromera velia and Phaeodactylum tricornutum. The method reported therefore may also be suitable for other still partially unexplored algae, such as cryptophytes.
Assuntos
Complexo de Proteína do Fotossistema I/metabolismo , Rodófitas/metabolismo , Clorofila/metabolismo , Espectrometria de Fluorescência , Xantofilas/metabolismoRESUMO
Photoprotective non-photochemical quenching (NPQ) represents an effective way to dissipate the light energy absorbed in excess by most phototrophs. It is often claimed that NPQ formation/relaxation kinetics are determined by xanthophyll composition. We, however, found that, for the alveolate alga Chromera velia, this is not the case. In the present paper, we investigated the reasons for the constitutive high rate of quenching displayed by the alga by comparing its light harvesting strategies with those of a model phototroph, the land plant Spinacia oleracea. Experimental results and in silico studies support the idea that fast quenching is due not to xanthophylls, but to intrinsic properties of the Chromera light harvesting complex (CLH) protein, related to amino acid composition and protein folding. The pKa for CLH quenching was shifted by 0.5 units to a higher pH compared with higher plant antennas (light harvesting complex II; LHCII). We conclude that, whilst higher plant LHCIIs are better suited for light harvesting, CLHs are 'natural quenchers' ready to switch into a dissipative state. We propose that organisms with antenna proteins intrinsically more sensitive to protons, such as C. velia, carry a relatively high concentration of violaxanthin to improve their light harvesting. In contrast, higher plants need less violaxanthin per chlorophyll because LHCII proteins are more efficient light harvesters and instead require co-factors such as zeaxanthin and PsbS to accelerate and enhance quenching.
Assuntos
Alveolados/fisiologia , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese , Prótons , Spinacia oleracea/fisiologia , Proteínas de Algas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
It has previously been shown that the long-term treatment of Arabidopsis thaliana with the chloroplast inhibitor lincomycin leads to photosynthetic membranes enriched in antennas, strongly reduced in photosystem II reaction centers (PSII) and with enhanced nonphotochemical quenching (NPQ) (Belgio et al. Biophys J 102:2761-2771, 2012). Here, a similar physiological response was found in the microalga Chromera velia grown under high light (HL). In comparison to cells acclimated to low light, HL cells displayed a severe re-organization of the photosynthetic membrane characterized by (1) a reduction of PSII but similar antenna content; (2) partial uncoupling of antennas from PSII; (3) enhanced NPQ. The decrease in the number of PSII represents a rather unusual acclimation response compared to other phototrophs, where a smaller PSII antenna size is more commonly found under high light. Despite the diminished PSII content, no net damage could be detected on the basis of the Photosynthesis versus irradiance curve and electron transport rates pointing at the excess capacity of PSII. We therefore concluded that the photoinhibition is minimized under high light by a lower PSII content and that cells are protected by NPQ in the antennas.
Assuntos
Aclimatação/efeitos da radiação , Alveolados/fisiologia , Alveolados/efeitos da radiação , Luz , Processos Fotoquímicos/efeitos da radiação , Alveolados/citologia , Clorofila/metabolismo , Clorofila A , Fluorescência , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/metabolismo , SolubilidadeRESUMO
It is often suggested that Life may lay outside the normal laws of Physics and particularly of Thermodynamics, though this point of view is refuted by many. As the Living State may be thought of as an open system, often far from equilibrium, most attempts at placing Life under the umbrella of the laws of Physics have been based, particularly in recent years, on non-equilibrium Thermodynamics and particularly the Maximum Entropy Production Principle. In this view it is the dissipation of entropy (heat) which permits the ever increasing complexity of Living Systems in biological evolution and the maintenance of this complexity. However, these studies usually consider such biological entities as whole cells, organs, whole organisms and even Life itself at the entire terrestrial level. This requires making assumptions concerning the Living State, which are often not soundly based on observation and lack a defined model structure. The present study is based on an entirely different approach, in which a classical thermodynamic analysis of a well-defined biological nanoparticle, plant Photosystem I, is performed. This photosynthetic structure, which absorbs light and performs primary and secondary charge separation, operates with a quantum efficiency close to one. It is demonstrated that when monochromatic light is absorbed by the lowest lying electronic transition, the chlorophyll Qy transition, entropy production in the system bath plus entropy changes internal to the system are numerically less than the entropy decrease of the light field. A Second Law violation is therefore suggested for these experimental conditions. This conclusion, while at first sight is supportive of the famous and much discussed statement of Schroedinger, that "Life feeds on negentropy", is analysed and the conditions in which this statement may be considered valid for a Plant Photosystem are defined and delimited. The remarkably high quantum efficiency, leading to minimal entropy production (energy wastage), seems to suggest that evolution of Photosystem I has gone down the road of maximal energy efficiency as distinct from maximal entropy production. Photosystem I cannot be considered a maximum entropy dissipation structure.
Assuntos
Clorofila/química , Entropia , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema II/química , Absorção FisiológicaRESUMO
In the present work, we report the first comparative spectroscopic investigation between Photosystem I (PSI) complexes isolated from two red clade algae. Excitation energy transfer was measured in PSI from Chromera velia, an alga possessing a split PsaA protein, and from the model diatom Phaeodactylum tricornutum. In both cases, the estimated effective photochemical trapping time was in the 15-25ps range, i.e. twice as fast as higher plants. In contrast to green phototrophs, the trapping time was rather constant across the whole emission spectrum. The weak wavelength dependence was attributed to the limited presence of long-wavelength emitting chlorophylls, as verified by low temperature spectroscopy. As the trapping kinetics of C. velia PSI were barely distinguishable from those of P. tricornutum PSI, it was concluded that the scission of PsaA protein had no significant impact on the overall PSI functionality. In conclusion, the two red clade algae analysed here, carried amongst the most efficient charge separation so far reported for isolated Photosystems.
Assuntos
Alveolados/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Rodófitas/metabolismo , Clorofila/metabolismo , Diatomáceas/metabolismo , Transferência de Energia/fisiologia , Cinética , Complexos de Proteínas Captadores de Luz/metabolismo , Espectrometria de FluorescênciaRESUMO
Aggregation induced conformational change of light harvesting antenna complexes is believed to constitute one of the pathways through which photosynthetic organisms can safely dissipate the surplus of energy while exposed to saturating light. In this study, Stark fluorescence (SF) spectroscopy is applied to minor antenna complexes (CP24, CP26 and CP29) both in their light-harvesting and energy-dissipating states to trace and characterize different species generated upon energy dissipation through aggregation (in-vitro) induced conformational change. SF spectroscopy could identify three spectral species in the dissipative state of CP24, two in CP26 and only one in CP29. The comprehensive analysis of the SF spectra yielded different sets of molecular parameters for the multiple spectral species identified in CP24 or CP26, indicating the involvement of different pigments in their formation. Interestingly, a species giving emission around the 730nm spectral region is found to form in both CP24 and CP26 following transition to the energy dissipative state, but not in CP29. The SF analyses revealed that the far red species has exceptionally large charge transfer (CT) character in the excited state. Moreover, the far red species was found to be formed invariably in both Zeaxanthin (Z)- and Violaxathin (V)-enriched CP24 and CP26 antennas with identical CT character but with larger emission yield in Z-enriched ones. This suggests that the carotenoid Z is not directly involved but only confers an allosteric effect on the formation of the far red species. Similar far red species with remarkably large CT character were also observed in the dissipative state of the major light harvesting antenna (LHCII) of plants [Wahadoszamen et al. PCCP, 2012], the fucoxanthin-chlorophyll protein (FCP) of brown algae [Wahadoszamen et al. BBA, 2014] and cyanobacterial IsiA [Wahadoszamen et al. BBA, 2015], thus pointing to identical sites and pigments active in the formation of the far red quenching species in different organisms.
Assuntos
Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese , Spinacia oleracea/metabolismo , Clorofila/efeitos da radiação , Transferência de Energia , Luz , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/efeitos da radiação , Fotossíntese/efeitos da radiação , Conformação Proteica , Especificidade da Espécie , Espectrometria de Fluorescência , Spinacia oleracea/química , Spinacia oleracea/efeitos da radiação , Relação Estrutura-Atividade , Xantofilas/metabolismo , Zeaxantinas/metabolismoRESUMO
A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction center subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25% of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress.
RESUMO
Non-photochemical quenching (NPQ) is a photoprotective mechanism in light-harvesting antennae. NPQ is triggered by chloroplast thylakoid lumen acidification and is accompanied by violaxanthin de-epoxidation to zeaxanthin, which further stimulates NPQ. In the present study, we show that violaxanthin can act in the opposite direction to zeaxanthin because an increase in the concentration of violaxanthin reduced NPQ in the light-harvesting antennae of Chromera velia. The correlation overlapped with a similar relationship between violaxanthin and NPQ as observed in isolated higher plant light-harvesting complex II. The data suggest that violaxanthin in C. velia can act as an inhibitor of NPQ, indicating that violaxanthin has to be removed from the vicinity of the protein to reach maximal NPQ.
Assuntos
Alveolados/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Processos Fotoquímicos , Alveolados/citologia , Alveolados/efeitos da radiação , Clorofila/metabolismo , Fluorescência , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Fatores de Tempo , Xantofilas/metabolismoRESUMO
Plants with varying levels of PsbS protein were grown on lincomycin. Enhanced levels of non-photochemical fluorescence quenching (NPQ) in over-expressers of the protein have been observed. This was accompanied by increased amplitude of the irreversible NPQ component, qI, previously considered to reflect mainly photoinhibition of PSII reaction centres (RCII). However, since RCIIs were largely absent the observed qI is likely to originate from the LHCII antenna. In chloroplasts of over-expressers of PsbS grown on lincomycin an abnormally large NPQ (â¼7) was characterised by a 0.34 ns average chlorophyll fluorescence lifetime. Yet the lifetime in the Fm state was similar to that of wild-type plants. 77K fluorescence emission spectra revealed a specific 700 nm peak typical of LHCII aggregates as well as quenching of the PSI fluorescence at 730 nm. The aggregated state manifested itself as a clear change in the distance between LHCII complexes detected by freeze-fracture electron microscopy. Grana thylakoids in the quenched state revealed 3 times more aggregated LHCII particles compared to the dark-adapted state. Overall, the results directly demonstrate the importance of LHCII aggregation in the NPQ mechanism and show that the PSII supercomplex structure plays no role in formation of the observed quenching.
Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/efeitos dos fármacos , Técnicas de Inativação de Genes , Complexos de Proteínas Captadores de Luz/deficiência , Complexos de Proteínas Captadores de Luz/genética , Lincomicina/farmacologia , Complexo de Proteína do Fotossistema II/genética , Espectrometria de Fluorescência , TemperaturaRESUMO
The light-harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures - stacks of dozens of thylakoids or supergrana. Freeze-fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light-harvesting membranes, organized into three-dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self-assembly of the three-dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.
Assuntos
Arabidopsis/ultraestrutura , Cloroplastos/ultraestrutura , Complexos de Proteínas Captadores de Luz/ultraestrutura , Complexo de Proteína do Fotossistema II/ultraestrutura , Viridiplantae/ultraestrutura , Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Fluorescência , Luz , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Prótons , Viridiplantae/metabolismoRESUMO
Arabidopsis plants grown at low light were exposed to a gradually increasing actinic light routine. This method allows for the discerning of the photoprotective component of NPQ, pNPQ and photoinhibition. They exhibited lower values of Photosystem II (PSII) yield in comparison to high-light grown plants, and higher calculated dark fluorescence level (F'o calc.) than the measured one (F'o act.). As a result, in low-light grown plants, the values of qP measured in the dark appeared higher than 1. Normally, F'o act. and F'o calc. match well at moderate light intensities but F'o act. becomes higher at increasing intensities due to reaction centre (RCII) damage; this indicates the onset of photoinhibition. To explain the unusual increase of qP in the dark in low-light grown plants, we have undertaken an analysis of PSII antenna size using biochemical and spectroscopic approaches. Sucrose gradient separation of thylakoid membrane complexes and fast fluorescence induction experiments illustrated that the relative PSII cross section does not increase appreciably with the rise in PSII antenna size in the low-light grown plants. This suggests that part of the increased LHCII antenna is less efficiently coupled to the RCII. A model based upon the existence of an uncoupled population LHCII is proposed to explain the discrepancies in calculated and measured values of F'o.
Assuntos
Aclimatação , Arabidopsis/fisiologia , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/efeitos da radiação , Clorofila/metabolismo , Cloroplastos/metabolismo , Fluorescência , Luz , Fotossíntese , Tilacoides/metabolismoRESUMO
The efficiency of protective energy dissipation by non-photochemical quenching (NPQ) in photosystem II (PSII) has been recently quantified by a new non-invasive photochemical quenching parameter, qPd. PSII yield (ФPSII) was expressed in terms of NPQ, and the extent of damage to the reaction centres (RCIIs) was calculated via qPd as: ФPSII=qPd×(F v/F m)/{1+[1-(F v/F m)]×NPQ}. Here this approach was used to determine the amount of NPQ required to protect all PSII reaction centres (pNPQ) under a gradually increasing light intensity, in the zeaxanthin-deficient (npq1) Arabidopsis mutant, compared with PsbS protein-deficient (npq4) and wild-type plants. The relationship between maximum pNPQ and tolerated light intensity for all plant genotypes followed similar trends. These results suggest that under a gradually increasing light intensity, where pNPQ is allowed to develop, it is only the amplitude of pNPQ which is the determining factor for protection. However, the use of a sudden constant high light exposure routine revealed that the presence of PsbS, not zeaxanthin, offered better protection for PSII. This was attributed to a slower development of pNPQ in plants lacking PsbS in comparison with plants that lacked zeaxanthin. This research adds further support to the value of pNPQ and qPd as effective parameters for assessing NPQ effectiveness in different types of plants.
Assuntos
Arabidopsis/metabolismo , Complexos de Proteínas Captadores de Luz/deficiência , Zeaxantinas/deficiência , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Luz , Complexos de Proteínas Captadores de Luz/genética , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiaçãoRESUMO
The light-harvesting antenna of higher plant photosystem II has an intrinsic capability for self-defence against intense sunlight. The thermal dissipation of excess energy can be measured as the non-photochemical quenching of chlorophyll fluorescence. It has recently been proposed that the transition between the light-harvesting and self-defensive modes is associated with a reorganization of light-harvesting complexes. Here we show that despite structural changes, the photosystem II cross-section does not decrease. Our study reveals that the efficiency of energy trapping by the non-photochemical quencher(s) is lower than the efficiency of energy capture by the reaction centres. Consequently, the photoprotective mechanism works effectively for closed rather than open centres. This type of defence preserves the exceptional efficiency of electron transport in a broad range of light intensities, simultaneously ensuring high photosynthetic productivity and, under hazardous light conditions, sufficient photoprotection for both the reaction centre and the light-harvesting pigments of the antenna.
Assuntos
Arabidopsis/fisiologia , Complexos de Proteínas Captadores de Luz/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Energia Solar , Modelos Biológicos , Fotossíntese/fisiologia , Folhas de Planta/fisiologiaRESUMO
The principle of quantifying the efficiency of protection of photosystem II (PSII) reaction centres against photoinhibition by non-photochemical energy dissipation (NPQ) has been recently introduced by Ruban & Murchie (2012 Biochim. Biophys. Acta 1817, 977-982 (doi:10.1016/j.bbabio.2012.03.026)). This is based upon the assessment of two key parameters: (i) the relationship between the PSII yield and NPQ, and (ii) the fraction of intact PSII reaction centres in the dark after illumination. In this paper, we have quantified the relationship between the amplitude of NPQ and the light intensity at which all PSII reaction centres remain intact for plants with different levels of PsbS protein, known to play a key role in the process. It was found that the same, nearly linear, relationship exists between the levels of the protective NPQ component (pNPQ) and the tolerated light intensity in all types of studied plants. This approach allowed for the quantification of the maximum tolerated light intensity, the light intensity at which all plant leaves become photoinhibited, the fraction of (most likely) unnecessary or 'wasteful' NPQ, and the fraction of photoinhibited PSII reaction centres under conditions of prolonged illumination by full sunlight. It was concluded that the governing factors in the photoprotection of PSII are the level and rate of protective pNPQ formation, which are often in discord with the amplitude of the conventional measure of photoprotection, the quickly reversible NPQ component, qE. Hence, we recommend pNPQ as a more informative and less ambiguous parameter than qE, as it reflects the effectiveness and limitations of the major photoprotective process of the photosynthetic membrane.
Assuntos
Cloroplastos/fisiologia , Metabolismo Energético/efeitos da radiação , Luz , Modelos Biológicos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Fenômenos Fisiológicos Vegetais , Arabidopsis , Fluorescência , Xantofilas/metabolismoRESUMO
Here we show how the protein environment in terms of detergent concentration/protein aggregation state, affects the sensitivity to pH of isolated, native LHCII, in terms of chlorophyll fluorescence quenching. Three detergent concentrations (200, 20 and 6µM n-dodecyl ß-d-maltoside) have been tested. It was found that at the detergent concentration of 6µM, low pH quenching of LHCII is close to the physiological response to lumen acidification possessing pK of 5.5. The analysis has been conducted both using arbitrary PAM fluorimetry measurements and chlorophyll fluorescence lifetime component analysis. The second led to the conclusion that the 3.5ns component lifetime corresponds to an unnatural state of LHCII, induced by the detergent used for solubilising the protein, whilst the 2ns component is rather the most representative lifetime component of the conformational state of LHCII in the natural thylakoid membrane environment when the non-photochemical quenching (NPQ) was absent. The 2ns component is related to a pre-aggregated LHCII that makes it more sensitive to pH than the trimeric LHCII with the dominating 3.5ns lifetime component. The pre-aggregated LHCII displayed both a faster response to protons and a shift in the pK for quenching to higher values, from 4.2 to 4.9. We concluded that environmental factors like lipids, zeaxanthin and PsbS protein that modulate NPQ in vivo could control the state of LHCII aggregation in the dark that makes it more or less sensitive to the lumen acidification. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
Assuntos
Clorofila/química , Complexos de Proteínas Captadores de Luz/química , Detergentes/farmacologia , Fluorescência , Concentração de Íons de Hidrogênio , Complexo de Proteína do Fotossistema II/química , Conformação Proteica , Multimerização ProteicaRESUMO
Light-harvesting pigment-protein complexes of photosystem II of plants have a dual function: they efficiently use absorbed energy for photosynthesis at limiting sunlight intensity and dissipate the excess energy at saturating intensity for photoprotection. Recent single-molecule spectroscopy studies on the trimeric LHCII complex showed that environmental control of the intrinsic protein disorder could in principle explain the switch between their light-harvesting and photoprotective conformations in vivo. However, the validity of this proposal depends strongly on the specificity of the protein dynamics. Here, a similar study has been performed on the minor monomeric antenna complexes of photosystem II (CP29, CP26, and CP24). Despite their high structural homology, similar pigment content and organization compared to LHCII trimers, the environmental response of these proteins was found to be rather distinct. A much larger proportion of the minor antenna complexes were present in permanently weakly fluorescent states under most conditions used; however, unlike LHCII trimers the distribution of the single-molecule population between the strongly and weakly fluorescent states showed no significant sensitivity to low pH, zeaxanthin, or low detergent conditions. The results support a unique role for LHCII trimers in the regulation of light harvesting by controlled fluorescence blinking and suggest that any contribution of the minor antenna complexes to photoprotection would probably involve a distinct mechanism.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Luz , Plantas/metabolismo , Plantas/efeitos da radiação , Meio Ambiente , Multimerização Proteica , Estrutura Quaternária de Proteína , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Apoproteínas/química , Complexos de Proteínas Captadores de Luz/metabolismo , Substituição de Aminoácidos , Apoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Luz , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/genética , Folhas de Planta/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Fluorescência , Temperatura , Xantofilas/química , Xantofilas/metabolismoRESUMO
The maximum chlorophyll fluorescence lifetime in isolated photosystem II (PSII) light-harvesting complex (LHCII) antenna is 4 ns; however, it is quenched to 2 ns in intact thylakoid membranes when PSII reaction centers (RCIIs) are closed (Fm). It has been proposed that the closed state of RCIIs is responsible for the quenching. We investigated this proposal using a new, to our knowledge, model system in which the concentration of RCIIs was highly reduced within the thylakoid membrane. The system was developed in Arabidopsis thaliana plants under long-term treatment with lincomycin, a chloroplast protein synthesis inhibitor. The treatment led to 1), a decreased concentration of RCIIs to 10% of the control level and, interestingly, an increased antenna component; 2), an average reduction in the yield of photochemistry to 0.2; and 3), an increased nonphotochemical chlorophyll fluorescence quenching (NPQ). Despite these changes, the average fluorescence lifetimes measured in Fm and Fm' (with NPQ) states were nearly identical to those obtained from the control. A 77 K fluorescence spectrum analysis of treated PSII membranes showed the typical features of preaggregation of LHCII, indicating that the state of LHCII antenna in the dark-adapted photosynthetic membrane is sufficient to determine the 2 ns Fm lifetime. Therefore, we conclude that the closed RCs do not cause quenching of excitation in the PSII antenna, and play no role in the formation of NPQ.