Chlorophyll fluorescence quenching in the alga Euglena gracilis

When far red light preincubated cells of Euglena gracilis are transferred to dark or light, chlorophyll fluorescence (F₀ and F_m) decreases. Non-photochemical quenching in the dark is suggested to be induced partly by chlororespiration and partly by changes in the distribution of excitation energy between the photosystems. Depending on the light intensities it was possible to resolve the non-photochemical quenching into at least three different components. The slowest relaxation phase of non-photochemical quenching occurred only after exposure to high light and was assigned to photoinhibition. The other two components were an energy-dependent quenching (qE), and the one which we attribute to a spill over mechanism. We suggest that both photosystems use a common antenna system consisting of LHC I and LHC II proteins. In contrast to higher plants, qE in Euglena gracilis is independent of the xanthophyll cycle and an aggregation of LHC II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.Abbreviations CAB chlorophyll a/b-binding - Chl chlorophyll - F_V variable fluorescence - IML intermittent light - LHC light harvesting complex - PFD photon flux density - qP photochemical quenching of chlorophyll fluoresence - qN non-photochemical quenching - qE energy-dependent quenching - qI photoinhibitory quenching - qT quenching by state transition     

Polyamines induce aggregation of LHC II and quenching of fluorescence in vitro

Dissipation of excess excitation energy within the light-harvesting complex of Photosystem II (LHC II) is a main process in plants, which is measured as the non-photochemical quenching of chlorophyll fluorescence or qE. We showed in previous works that polyamines stimulate qE in higher plants in vivo and in eukaryotic algae in vitro. In the present contribution we have tested whether polyamines can stimulate quenching in trimeric LHC II and monomeric light-harvesting complex b proteins from higher plants. The tetramine spermine was the most potent quencher and induced aggregation of LHC II trimers, due to its highly cationic character. Two transients are evident at 100μM and 350μM for the fluorescence and absorbance signals of LHC II respectively. On the basis of observations within this work, some links between polyamines and the activation of qE in vivo is discussed.     

Photoprotective energy quenching in the red alga Porphyridium purpureum occurs at the core antenna of the photosystem II but not at its reaction center

Photosynthetic organisms have evolved light-harvesting antennae over time. In cyanobacteria, external phycobilisomes (PBSs) are the dominant antennae, whereas in green algae and higher plants, PBSs have been replaced by proteins of the Lhc family that are integrated in the membrane. Red algae represent an evolutionary intermediate between these two systems, as they employ both PBSs and membrane LHCR proteins as light-harvesting units. Understanding how red algae cope with light is not only interesting for biotechnological applications, but is also of evolutionary interest. For example, energy-dependent quenching (qE) is an essential photoprotective mechanism widely used by species from cyanobacteria to higher plants to avoid light damage; however, the quenching mechanism in red algae remains largely unexplored. Here, we used both pulse amplitude-modulated (PAM) and time-resolved chlorophyll fluorescence to characterize qE kinetics in the red alga Porphyridium purpureum. PAM traces confirmed that qE in P. purpureum is activated by a decrease in the thylakoid lumen pH, whereas time-resolved fluorescence results further revealed the quenching site and ultrafast quenching kinetics. We found that quenching exclusively takes place in the photosystem II (PSII) complexes and preferentially occurs at PSII’s core antenna rather than at its reaction center, with an overall quenching rate of 17.6 ± 3.0 ns⁻¹. In conclusion, we propose that qE in red algae is not a reaction center type of quenching, and that there might be a membrane-bound protein that resembles PsbS of higher plants or LHCSR of green algae that senses low luminal pH and triggers qE in red algae.     

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679?nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.     

The transiently generated nonphotochemical quenching of excitation energy in Arabidopsis leaves is modulated by zeaxanthin

Upon the transition of dark-adapted plants to low light, the energy-dependent quenching (qE) of excitation energy is only transiently induced due to the only transient generation of the transthylakoid pH gradient. We investigated the transient qE (qE(TR)) in different Arabidopsis (Arabidopsis thaliana) mutants. In dark-adapted plants, qE(TR) was absent in the npq4 mutant (deficient in the PsbS protein) and the pgr1 mutant (restricted in lumen acidification). In comparison with wild-type plants, qE(TR) was reduced in the zeaxanthin (Zx)-deficient npq1 mutant and increased in the Zx-accumulating npq2 mutant. After preillumination of plants (to allow the synthesis of large amounts of Zx), the formation and relaxation of qE(TR) was accelerated in all plants (except for npq4) in comparison with the respective dark-adapted plants. The extent of qE(TR), however, was unchanged in npq1 and npq4, decreased in npq2, but increased in wild-type and pgr1 plants. Even in presence of high levels of Zx, qE(TR) in pgr1 mutants was still lower than that in wild-type plants. In the presence of the uncoupler nigericin, qE(TR) was completely abolished in all genotypes. Thus, the transient qE(TR) shows essentially the same characteristics as the steady-state qE; it is strictly dependent on the PsbS protein and a low lumen pH, but the extent of qE(TR) is largely modulated by Zx. These results indicate that qE(TR) does not represent a different quenching mechanism in comparison with the steady-state qE, but simply reflects the response of qE to the dynamics of the lumen pH during light activation of photosynthesis.     

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.     

Models and measurements of energy-dependent quenching

Energy-dependent quenching (qE) in photosystem II (PSII) is a pH-dependent response that enables plants to regulate light harvesting in response to rapid fluctuations in light intensity. In this review, we aim to provide a physical picture for understanding the interplay between the triggering of qE by a pH gradient across the thylakoid membrane and subsequent changes in PSII. We discuss how these changes alter the energy transfer network of chlorophyll in the grana membrane and allow it to switch between an unquenched and quenched state. Within this conceptual framework, we describe the biochemical and spectroscopic measurements and models that have been used to understand the mechanism of qE in plants with a focus on measurements of samples that perform qE in response to light. In addition, we address the outstanding questions and challenges in the field. One of the current challenges in gaining a full understanding of qE is the difficulty in simultaneously measuring both the photophysical mechanism of quenching and the physiological state of the thylakoid membrane. We suggest that new experimental and modeling efforts that can monitor the many processes that occur on multiple timescales and length scales will be important for elucidating the quantitative details of the mechanism of qE.     

Is PsbS the site of non-photochemical quenching in photosynthesis?

The PsbS protein of photosystem II functions in the regulation of photosynthetic light harvesting. Along with a low thylakoid lumen pH and the presence of de-epoxidized xanthophylls, PsbS is necessary for photoprotective thermal dissipation (qE) of excess absorbed light energy in plants, measured as non-photochemical quenching of chlorophyll fluorescence. What is known about PsbS in relation to the hypothesis that this protein is the site of qE is reviewed here.     

Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced DeltapH

Variations in the light environment require higher plants to regulate the light harvesting process. Under high light a mechanism known as non-photochemical quenching (NPQ) is triggered to dissipate excess absorbed light energy within the photosystem II (PSII) antenna as heat, preventing photodamage to the reaction center. The major component of NPQ, known as qE, is rapidly reversible in the dark and dependent upon the transmembrane proton gradient (ΔpH), formed as a result of photosynthetic electron transport. Using diaminodurene and phenazine metasulfate, mediators of cyclic electron flow around photosystem I, to enhance ΔpH, it is demonstrated that rapidly reversible qE-type quenching can be observed in intact chloroplasts from Arabidopsis plants lacking the PsbS protein, previously believed to be indispensible for the process. The qE in chloroplasts lacking PsbS significantly quenched the level of fluorescence when all PSII reaction centers were in the open state (F(o) state), protected PSII reaction centers from photoinhibition, was modulated by zeaxanthin and was accompanied by the qE-typical absorption spectral changes, known as ΔA(535). Titrations of the ΔpH dependence of qE in the absence of PsbS reveal that this protein affects the cooperativity and sensitivity of the photoprotective process to protons. The roles of PsbS and zeaxanthin are discussed in light of their involvement in the control of the proton-antenna association constant, pK, via regulation of the interconnected phenomena of PSII antenna reorganization/aggregation and hydrophobicity.     

Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum

Photosynthetic organisms in nature often experience light fluctuations. While low light conditions limit the energy uptake by algae, light absorption exceeding the maximal rate of photosynthesis may go along with enhanced formation of potentially toxic reactive oxygen species. To preempt high light-induced photodamage, photosynthetic organisms evolved numerous photoprotective mechanisms. Among these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to dissipate thermally the excessively absorbed energy. Diatoms thrive in all aquatic environments and thus belong to the most important primary producers on earth. qE in diatoms is provided by a concerted action of Lhcx proteins and the xanthophyll cycle pigment diatoxanthin. While the exact Lhcx activation mechanism of diatom qE is unknown, two lumen-exposed acidic amino acids within Lhcx proteins were proposed to function as regulatory switches upon light-induced lumenal acidification. By introducing a modified Lhcx1 lacking these amino acids into a Phaeodactylum tricornutum Lhcx1-null qE knockout line, we demonstrate that qE is unaffected by these two amino acids. Based on sequence comparisons with Lhcx4, being incapable of providing qE, we perform domain swap experiments of Lhcx4 with Lhcx1 and identify two peptide motifs involved in conferring qE. Within one of these motifs, we identify a tryptophan residue with a major influence on qE establishment. This tryptophan residue is located in close proximity to the diadinoxanthin/diatoxanthin-binding site based on the recently revealed diatom Lhc crystal structure. Our findings provide a structural explanation for the intimate link of Lhcx and diatoxanthin in providing qE in diatoms.     

The chloroplast NADPH thioredoxin reductase C,NTRC, controls non‐photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis

High irradiances may lead to photooxidative stress in plants, and non‐photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light‐limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans‐thylakoid ΔpH and have 10‐fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc–psbs double‐knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc–psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield.     

Disentangling the low-energy states of the major light-harvesting complex of plants and their role in photoprotection

The ability to dissipate large fractions of their absorbed light energy as heat is a vital photoprotective function of the peripheral light-harvesting pigment–protein complexes in photosystem II of plants. The major component of this process, known as qE, is characterised by the appearance of low-energy (red-shifted) absorption and fluorescence bands. Although the appearance of these red states has been established, the molecular mechanism, their site and particularly their involvement in qE are strongly debated. Here, room-temperature single-molecule fluorescence spectroscopy was used to study the red emission states of the major plant light-harvesting complex (LHCII) in different environments, in particular conditions mimicking qE. It was found that most states correspond to peak emission at around 700 nm and are unrelated to energy dissipative states, though their frequency of occurrence increased under conditions that mimicked qE. Longer-wavelength emission appeared to be directly related to energy dissipative states, in particular emission beyond 770 nm. The ensemble average of the red emission bands shares many properties with those obtained from previous bulk in vitro and in vivo studies. We propose the existence of at least three excitation energy dissipating mechanisms in LHCII, each of which is associated with a different spectral signature and whose contribution to qE is determined by environmental control of protein conformational disorder. Emission at 700 nm is attributed to a conformational change in the Lut 2 domain, which is facilitated by the conformational change associated with the primary quenching mechanism involving Lut 1.     

Slow zeaxanthin accumulation and the enhancement of CP26 collectively contribute to an atypical non-photochemical quenching in macroalga Ulva prolifera under high light

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants, which dissipates excess energy and further protects the photosynthetic apparatus under high light stress. NPQ can be dissected into a number of components: qE, qZ, and qI. In general, NPQ is catalyzed by two independent mechanisms, with the faster-activated quenching catalyzed by the monomeric light-harvesting complex (LHCII) proteins and the slowly activated quenching catalyzed by LHCII trimers, both processes depending on zeaxanthin but to different extent. Here, we studied the NPQ of the intertidal green macroalga, Ulva prolifera, and found that the NPQ of U. prolifera lack the faster-activated quenching, and showed much greater sensitivity to dithiothreitol (DTT) than to dicyclohexylcarbodiimide (DCCD). Further results suggested that the monomeric LHC proteins in U. prolifera included only CP29 and CP26, but lacked CP24, unlike Arabidopsis thaliana and the moss Physcomitrella patens. Moreover, the expression levels of CP26 increased significantly following exposure to high light, but the concentrations of the two important photoprotective proteins (PsbS and light-harvesting complex stress-related [LhcSR]) did not change upon the same conditions. Analysis of the xanthophyll cycle pigments showed that, upon exposure to high light, zeaxanthin synthesis in U. prolifera was gradual and much slower than that in P. patens, and could effectively be inhibited by DTT. Based on these results, we speculate the enhancement of CP26 and slow zeaxanthin accumulation provide an atypical NPQ, making this green macroalga well adapted to the intertidal environments.     

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal DeltapH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1 approximately lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.     

Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants

Biochemical and physiological acclimation to different light environments is crucial for plant growth and survival. In high light (HL), feedback de-excitation (qE) is a well-known photoprotective mechanism that dissipates excess excitation energy in the light-harvesting antenna of photosystem II (PSII) and relieves excitation pressure in the photosynthetic electron transport chain. The xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but also have roles as antioxidants. Although several studies have shown that qE is important during short-term fluctuations in light intensity, here we show that it is not required for the growth of Arabidopsis thaliana in prolonged HL conditions in the laboratory. Mutants that are deficient in qE alone, qE and Z synthesis, or in qE, Z synthesis and also L synthesis were able to grow at 1800 micromol photons m(-2) s(-1) and exhibited no major symptoms of photooxidative stress. The mutants (and wild type) acclimated to HL by increasing photosynthetic capacity and decreasing light harvesting, which together rendered qE less important for photoprotection. At a metabolite level, the HL-grown mutants appeared to compensate for their remaining qE deficit with increased alpha-tocopherol and ascorbate levels compared to the wild type. The specificity of this response provides insight into the relationship between qE and the antioxidant network in plants.     

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal ΔpH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1≈lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.     

Allocation of absorbed light energy in PSII to thermal dissipations in the presence or absence of PsbS subunits of rice

The thermal dissipation (TD) of absorbed light energy in PSII is considered to be an important photoprotection process in photosynthesis. A major portion of TD has been visualized through the analysis of Chl fluorescence as energy quenching (qE) which depends on the presence of the PsbS subunit. Although the physiological importance of qE-associated TD (qE-TD) has been widely accepted, it is not yet clear how much of the absorbed light energy is dissipated through a qE-associated mechanism. In this study, the fates of absorbed light energy in PSII with regard to different TD processes, including qE-TD, were quantitatively estimated by the typical energy allocation models using transgenic rice in which psbS genes were silenced by RNA interference (RNAi). The silencing of psbS genes resulted in a decrease in the light-inducible portion of TD, whereas the allocation of energy to electron transport did not change over a wide range of light intensities. The allocation models indicate that the energy allocated to qE-TD under saturating light is 30-50%. We also showed that a large portion of absorbed light energy is thermally dissipated in manners that are independent of qE. The nature of such dissipations is discussed.