Non-photochemical Quenching Mechanism Research Articles

How much heat does non-photochemical quenching produce?

Non-photochemical quenching (NPQ) is a protective mechanism used by plants to safely dissipate excess absorbed light energy as heat, minimizing photo-oxidative damage. Although the importance of NPQ as a safety valve for photosynthesis is well-known, the physiological and environmental effects of the heat produced remain unclear because the amount of heat produced by NPQ is considered negligible, and its physiological effects have not been directly observed. Here, we calculated the heat produced by NPQ and evaluated its impact on the leaf and global warming based on simplified models. Our evaluation showed that the heat produced by NPQ in a given leaf area is 63.9 W m-2 under direct sunlight. Under the standard condition, NPQ warms up the leaf at less than 0.1°C, but it could be 1°C under particular conditions with low thermal conductance. We also estimated the thermal radiation of vegetation's NPQ to be 2.2 W m-2 par global averaged surface area. It is only 0.55% of the thermal radiation by the Earth's surface, but still significant in the current climate change response. We further discuss the possible function of NPQ to plant physiology besides the safety valve and provide strategies with artificial modification of the NPQ mechanism to increase food production and mitigate global warming.

Chilling Upregulates Expression of the PsbS and LhcSR Genes in the Chloroplasts of the Green Microalga Lobosphaera incisa IPPAS C-2047.

Non-photochemical quenching (NPQ) of excited chlorophyll states is essential for protecting the photosynthetic apparatus (PSA) from the excessive light-induced damage in all groups of oxygenic photosynthetic organisms. The key component of the NPQ mechanism in green algae and some other groups of algae and mosses is the LhcSR protein of the light harvesting complex (LHC) protein superfamily. In vascular plants, LhcSR is replaced by PsbS, another member of the LHC superfamily and a subunit of photosystem II (PSII). PsbS also performs the photoprotective function in mosses. For a long time, PsbS had been believed to be nonfunctional in green algae, although the corresponding gene was discovered in the genome of these organisms. The first evidence of the PsbS accumulation in the model green alga Chlamydomonas reinhardtii in response to the increase in irradiance was obtained only six years ago. However, the observed increase in the PsbS content was short-termed (on an hour-timescale). Here, we report a significant (more than three orders of magnitude) and prolonged (four days) upregulation of PsbS expression in response to the chilling-induced high-light stress followed by a less significant (~ tenfold) increase in the PsbS expression for nine days. This is the first evidence for the long-term upregulation of the PsbS expression in green alga (Chlorophyta) in response to stress. Our data indicate that the role of PsbS in the PSA of Chlorophyta is not limited to the first-line defense against stress, as it was previously assumed, but includes full-scale participation in the photoprotection of PSA from the environmental stress factors.

Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein

The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09 ns (6% of unquenched brightness) and 0.21 ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.

Photosynthetic gas-exchange and PSII photochemical acclimation to drought in a native and non-native xerophytic species (Artemisia ordosica and Salix psammophila)

The mechanisms of plant photosynthetic acclimation to drought remains incomplete. We examined the photosynthetic performance through plant photosystem-fluorescence response to a long-lasting, anomalous summer drought (i.e., lasting for 61 days, with soil water content <0.1 m3·m−3) and subsequent post-drought recovery of a native and a non-native desert shrub species (Artemisia ordosica and Salix psammophila, respectively) growing in northwest China. Key indicators to the examination are derived estimates of plant stomatal conductance (gs), transpiration (Tr), water use efficiency (WUE), maximum and actual photochemical efficiency (Fv/Fm and ФPSII), non-photochemical quenching (NPQ), and leaf photosynthesis (PN) from simultaneous, continuous in situ measurements of gas exchange and chlorophyll fluorescence. Chlorophyll fluorescence-associated indicator “Fv/Fm” for both species was down regulated in response to deficits in soil water content (SWC), with differential SWC thresholds of 0.07 and 0.08 m3 m−3 for A. ordosica and S. psammophila, respectively. The results revealed that both species acclimate to summer drought by a stomatal-regulation mechanism of reducing gs and WUE and by a PSII NPQ mechanism of dissipating the excessive light energy, indicative of a water-conservation strategy in the acclimation to drought. In comparison with the non-native species, the native species generally had greater photosynthetic performance under water-deficit conditions demonstrating higher transpiration, net CO2 assimilation, and greater PSII photochemical efficiency. Lower WUE and greater drought resistance and resilience, combined with minor changes in PSII photochemical efficiency are consistent with a well-defined water-conservation response by A. ordosica. The results support the hypothesis that native desert-shrub species can potentially outperform non-native shrub species, due to stronger resistance and faster overall recovery from long-lasting drought. Therefore, with increased drought severity and duration anticipated with future climate change, the native shrub species is suggested to be used in desertification control and ecological restoration for sustainable ecosystem management.

Nitrogen starvation induces distinct photosynthetic responses and recovery dynamics in diatoms and prasinophytes.

Nitrogen stress is an important control on the growth of phytoplankton and varying responses to this common condition among taxa may affect their relative success within phytoplankton communities. We analyzed photosynthetic responses to nitrogen (N) stress in two classes of phytoplankton that often dominate their respective size ranges, diatoms and prasinophytes, selecting species of distinct niches within each class. Changes in photosynthetic structures appeared similar within each class during N stress, but photophysiological and growth responses were more species- or niche-specific. In the coastal diatom Thalassiosira pseudonana and the oceanic diatom T. weissflogii, N starvation induced large declines in photosynthetic pigments and Photosystem II (PSII) quantity and activity as well as increases in the effective absorption cross-section of PSII photochemistry (σʹPSII). These diatoms also increased photoprotection through energy-dependent non-photochemical quenching (NPQ) during N starvation. Resupply of N in diatoms caused rapid recovery of growth and relaxation of NPQ, while recovery of PSII photochemistry was slower. In contrast, the prasinophytes Micromonas sp., an Arctic Ocean species, and Ostreococcus tauri, a temperate coastal eutrophile, showed little change in photosynthetic pigments and structures and a decline or no change, respectively, in σʹPSII with N starvation. Growth and PSII function recovered quickly in Micromonas sp. after resupply of N while O. tauri failed to recover N-replete levels of electron transfer from PSII and growth, possibly due to their distinct photoprotective strategies. O. tauri induced energy-dependent NPQ for photoprotection that may suit its variable and nutrient-rich habitat. Micromonas sp. relies upon both energy-dependent NPQ and a sustained, energy-independent NPQ mechanism. A strategy in Micromonas sp. that permits photoprotection with little change in photosynthetic structures is consistent with its Arctic niche, where low temperatures and thus low biosynthetic rates create higher opportunity costs to rebuild photosynthetic structures.

A siphonous morphology affects light-harvesting modulation in the intertidal green macroalga Bryopsis corticulans (Ulvophyceae)

Main conclusionThe macroalga Bryopsis corticulans relies on a sustained protective NPQ and a peculiar body architecture to efficiently adapt to the extreme light changes of intertidal shores.During low tides, intertidal algae experience prolonged high light stress. Efficient dissipation of excess light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is therefore required to avoid photodamage. Light-harvesting regulation was studied in the intertidal macroalga Bryopsis corticulans, during high light and air exposure. Photosynthetic capacity and NPQ kinetics were assessed in different filament layers of the algal tufts and in intact chloroplasts to unravel the nature of NPQ in this siphonous green alga. We found that the morphology and pigment composition of the B. corticulans body provides functional segregation between surface sunlit filaments (protective state) and those that are underneath and undergo severe light attenuation (light-harvesting state). In the surface filaments, very high and sustained NPQ gradually formed. NPQ induction was triggered by the formation of transthylakoid proton gradient and independent of the xanthophyll cycle. PsbS and LHCSR proteins seem not to be active in the NPQ mechanism activated by this alga. Our results show that B. corticulans endures excess light energy pressure through a sustained protective NPQ, not related to photodamage, as revealed by the unusually quick restoration of photosystem II (PSII) function in the dark. This might suggest either the occurrence of transient PSII photoinactivation or a fast rate of PSII repair cycle.

Slow and Fast Fluorescence Quenching of LHCII in Chlamydomonas Reinhardtii Cells

In this work we have studied the process of non-photochemical quenching (NPQ) in vivo in the model green alga C. reinhardtii. NPQ is the process that protects algae and plants from high light damage by dissipating large part of the absorbed energy as heat and which is activated by the low lumenal pH. Despite a large research effort the NPQ mechanism remains debated. The main obstacle to understand NPQ resides in the gap between in vitro and in vivo studies. On the one hand, the complexity of the thylakoid membrane makes it very difficult to obtain molecular information from in vivo experiments. On the other hand, a good in vitro system for the study of the quenching is not available. This has generated to a series of contrasting models that cannot be validated. We have developed a “minimal NPQ cell” which allows us to study the effect of the individual NPQ players in the membrane of living cells. We show that LHCII, the main antenna complexes of algae and plants, exists in different quenching states in the membrane depending on the growth conditions. This difference is suggested to be due to a difference in crowding. However, we also show that LHCII is not able to switch to the quenched conformation in response to pH. Instead, the presence of a very small amount of the protein LHCSR1 is sufficient to induce a large quenching (50%) in a membrane that contains only LHCII when the pH drops to 5.5. The quenching is very fast and completely reversible. Time-resolved fluorescence measurements at room and low temperature show that the quenching occurs in 360 ps and is not induced by additional clustering of LHCII in the membrane. Based on these results we propose a new model that links NPQ to long term acclimation responses.

Possible role of interference, protein noise, and sink effects in nonphotochemical quenching in photosynthetic complexes.

We analyze theoretically a simple and consistent quantum mechanical model that reveals the possible role of quantum interference, protein noise, and sink effects in the nonphotochemical quenching (NPQ) in light-harvesting complexes (LHCs). The model consists of a network of five interconnected sites (excitonic states of light-sensitive molecules) responsible for the NPQ mechanism. The model also includes the "damaging" and the dissipative channels. The damaging channel is responsible for production of singlet oxygen and other destructive outcomes. In our model, both damaging and "dissipative" charge transfer channels are described by discrete electron energy levels attached to their sinks, that mimic the continuum part of electron energy spectrum. All five excitonic sites interact with the protein environment that is modeled using a stochastic process. Our approach allowed us to derive the exact and closed system of linear ordinary differential equations for the reduced density matrix and its first momentums. These equations are solved numerically including for strong interactions between the light-sensitive molecules and protein environment. As an example, we apply our model to demonstrate possible contributions of quantum interference, protein noise, and sink effects in the NPQ mechanism in the CP29 minor LHC. The numerical simulations show that using proper combination of quantum interference effects, properties of noise, and sinks, one can significantly suppress the damaging channel. Our findings demonstrate the possible role of interference, protein noise, and sink effects for modeling, engineering, and optimizing the performance of the NPQ processes in both natural and artificial light-harvesting complexes.

Distortions of the Xanthophylls Caused by Interactions with Neighboring Pigments and the LHCII Protein Are Crucial for Studying Energy Transfer Pathways within the Complex.

It has been proposed that photoprotective non-photochemical quenching (NPQ) in higher plants arises from a conformational change in the antenna which alters pigment-pigment interactions. This brings about the formation of energy quenching "traps" that capture and dissipate excitation energy as heat. We have used the semiempirical AM1-CAS-CI method combined with the transition density cube (TDC) approach to model chlorophyll (Chl) to xanthophyll (Xanth) resonant Coulomb couplings in the crystal structure of LHCII. Due to its proposed role as the NPQ quenching site we have focused on lutein interactions and have explored how distortions to lutein conformation, as well as interpigment distances and relative orientations, affect this coupling. Our calculations indicate that distortions as well as Chl-lutein angle have a significant effect on coupling, whereas interpigment distances have a relatively minor effect. We therefore conclude that particular attention to the distortions of the Xanths should be given for calculation of energy transfer pathways and study of the NPQ mechanism.

Superradiance Transition and Nonphotochemical Quenching in Photosynthetic Complexes

We demonstrate numerically that superradiance could play a significant role in nonphotochemical quenching (NPQ) in light-harvesting complexes. Our model consists of a network of five interconnected sites (discrete excitonic states) that are responsible for the NPQ mechanism. Damaging and charge transfer states are linked to their sinks (independent continuum electron spectra), in which the chemical reactions occur. The superradiance transition in the charge transfer (or in the damaging) channel, occurs at particular electron transfer rates from the discrete to the continuum electron spectra, and can be characterized by a segregation of the imaginary parts of the eigenvalues of the effective non-Hermitian Hamiltonian. All five excitonic sites interact with their protein environment that is modeled by a random stochastic process. We find the region of parameters in which the superradiance transition into the charge transfer channel takes place. We demonstrate that this superradiance transition has the capability of producing optimal NPQ performance.

Photoprotection in the green tidal alga Ulva prolifera: role of LHCSR and PsbS proteins in response to high light stress

Ulva prolifera, an intertidal macroalga, has to adapt to wide variations in light intensity, making this species particularly rewarding for studying the evolution of photoprotective mechanisms. Intense light induced increased non-photochemical quenching (NPQ) and stimulated de-epoxidation of xanthophyll cycle components, while DTT-treated samples had lower NPQ capacity, indicating that the xanthophyll cycle must participate in photoprotection. In this work, we found that the PsbS-related NPQ was maintained in U.prolifera. According to analysed gene expression, both LhcSR and psbS were up-regulated in high light, suggesting that these two genes are light-induced. LHCSR and PsbS proteins were present at different light intensities and accumulated under high light conditions, and PsbS concentrations were higher than LHCSR, showing that the NPQ mechanism of U.prolifera is more dependent on PsbS protein concentration. Moreover, the level of both LHCSR and PsbS proteins was high even in the darkness, and neither the transcript level nor protein content of LhcSR and psbS genes varied significantly following short-term exposure to intense light. These findings suggest that this alga can modulate NPQ levels through regulation of the xanthophyll cycle and concentrations of PsbS and/or LHCSR.

Investigation of OCP-triggered dissipation of excitation energy in PSI/PSII-less Synechocystis sp. PCC 6803 mutant using non-linear laser fluorimetry

In order to prevent photodestruction by high light, photosynthetic organisms have evolved a number of mechanisms, known as non-photochemical quenching (NPQ), that deactivate the excited states of light harvesting pigments. Here we investigate the NPQ mechanism in the cyanobacterium Synechocystis sp. PCC 6803 mutant deficient in both photosystems. Using non-linear laser fluorimetry, we have determined molecular photophysical characteristics of phycocyanin and spectrally distinct forms of allophycocyanin for the cells in non-quenched and quenched states. Our analysis of non-linear fluorescence characteristics revealed that NPQ activation leads to an ~2-fold decrease in the relaxation times of both allophycocyanin fluorescence components, F660 and F680, and a 5-fold decrease in the effective excitation cross-section of F680, suggesting an emergence of a pathway of energy dissipation for both types of allophycocyanin. In contrast, NPQ does not affect the rates of singlet–singlet exciton annihilation. This indicates that, upon NPQ activation, the excess excitation energy is transferred from allophycocyanins to quencher molecules (presumably 3′hydroxyechinenone in the orange carotenoid protein), rather than being dissipated due to conformational changes of chromophores within the phycobilisome core. Kinetic measurements of fluorescence quenching in the Synechocystis mutant revealed the presence of several stages in NPQ development, as previously observed in the wild type. However, the lack of photosystems in the mutant enhanced the magnitude of NPQ as compared to the wild type, and allowed us to better characterize this process. Our results suggest a more complex kinetics of the NPQ process, thus clarifying a multistep model for the formation of the quenching center.

THE IMPACT OF NONPHOTOCHEMICAL QUENCHING OF FLUORESCENCE ON THE PHOTON BALANCE IN DIATOMS UNDER DYNAMIC LIGHT CONDITIONS(1).

The nonphotochemical quenching (NPQ) of fluorescence is an important photoprotective mechanism in particular under dynamic light conditions. Its photoprotective potential was suggested to be a functional trait of algal diversity. In the present study, the influence of the photoprotective capacity on the growth balance was investigated in two diatoms, which possess different NPQ characteristics. It was hypothesized that under fluctuating light conditions Cyclotella meneghiniana Kütz. would benefit from its large and flexible NPQ potential, whereas the comparably small NPQ capacity in Skeletonema costatum (Grev.) Cleve should exert an unfavorable impact on growth. The results of the study clearly falsify this hypothesis. Although C. meneghiniana possesses a fast NPQ component, this diatom was not able to recover its full NPQ capacity under fluctuating light. On the other hand, the induction of NPQ at relatively low irradiance in S. costatum resulted in rather small differences in the fraction of energy dissipation by the NPQ mechanism in the comparison of both diatoms. Larger differences were found in the metabolic characteristics. Both diatoms differed in their biomass composition, with a higher content of lipids in C. meneghiniana but higher amounts of carbohydrates in S. costatum. Finally, the lower degree of reduction in the biomass compensated for the higher respiration rates in S. costatum and resulted in a higher quantum efficiency of biomass production. An indirect correlation between the photoprotective and the metabolic capacity is discussed.

Non-photochemical fluorescence quenching in Chromera velia is enabled by fast violaxanthin de-epoxidation

Non-photochemical quenching (NPQ) is a mechanism protecting photosynthetic organisms against excessive irradiation. Here, we analyze a unique NPQ mechanism in the alga Chromera velia, a recently discovered close relative of apicomplexan parasites. NPQ in C. velia is enabled by an operative and fast violaxanthin de-epoxidation to zeaxanthin without accumulation of antheraxanthin. In C. velia violaxanthin also serves as a main light-harvesting pigment. Therefore, in C. velia violaxanthin acts as a key factor in both light harvesting and photoprotection. This is in contrast to a similar alga, Nannochloropsis limnetica, where violaxanthin has only light-harvesting function.

Role of PSBS and LHCSR in Physcomitrella patens acclimation to high light and low temperature

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. One of them, non-photochemical quenching (NPQ), consists in the thermal dissipation of energy absorbed in excess. In vascular plants NPQ relies on the activity of PSBS, whereas in the green algae Chlamydomonas reinhardtii it requires a different protein, LHCSR. The moss Physcomitrella patens is the only known organism in which both proteins are present and active in triggering NPQ, making this organism particularly interesting for the characterization of this protection mechanism. We analysed the acclimation of Physcomitrella to high light and low temperature, finding that these conditions induce an increase in NPQ correlated to overexpression of both PSBS and LHCSR. Mutants depleted of PSBS and/or LHCSR showed that modulation of their accumulation indeed determines NPQ amplitude. All mutants with impaired NPQ also showed enhanced photosensitivity when exposed to high light or low temperature, indicating that in this moss the fast-responding NPQ mechanism is also involved in long-term acclimation.

Interactions between the Photosystem II Subunit PsbS and Xanthophylls Studied in Vivo and in Vitro

The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.

Mechanistic aspects of xanthophyll cycle‐dependent photoprotection in higher plant chloroplasts and leaves

Higher plants must dissipate absorbed light energy that exceeds the photosynthetic capacity to avoid molecular damage to the pigments and proteins that comprise the photosynthetic apparatus. Described in this minireview is a current view of the biochemical, biophysical and bioenergetic aspects of the primary photoprotective mechanism responsible for dissipating excess excitation energy as heat from photosystem II (PSII). The photoprotective heat dissipation is measured as nonphotochemical quenching (NPQ) of the PSII chlorophyll a (Chl a) fluorescence. The NPQ mechanism is controlled by the trans‐thylakoid membrane pH gradient (ΔpH) and the special xanthophyll cycle pigments. In the NPQ mechanism, the de‐epoxidized endgroup moieties and the trans‐thylakoid membrane orientations of antheraxanthin (A) and zeaxanthin (Z) strongly affect their interactions with protonated chlorophyll binding proteins (CPs) of the PSII inner antenna. The CP protonation sites and steps are influenced by proton domains sequestered within the proteo‐lipid core of the thylakoid membrane. Xanthophyll cycle enrichment around the CPs may explain why changes in the peripheral PSII antenna size do not necessarily affect either the concentration of the xanthophyll cycle pigments on a per PSII unit basis or the NPQ mechanism. Recent time‐resolved PSII Chi a fluorescence studies suggest the NPQ mechanism switches PSII units to an increased rate constant of heat dissipation in a series of steps that include xanthophyll de‐epoxidation, CP‐protonation and binding of the xanthophylls to the protonated CPs; the concerted process can be described with a simple two‐step, pH‐activation model. The xanthophyll cycle‐dependent NPQ mechanism is profoundly influenced by temperatures suboptimal for photosynthesis via their effects on the trans‐thylakoid membrane energy coupling system. Further, low temperature effects can be grouped into either short term (minutes to hours) or long term (days to seasonal) series of changes in the content and composition of the PSII pigment‐proteins. This minireview concludes by briefly highlighting primary areas of future research interest regarding the NPQ mechanism.