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Balancing the central roles of the thylakoid proton gradient
Abstract
The photosynthetic electron transfer chain generates proton motive force (pmf), composed of both electric field (Deltapsi) and concentration (DeltapH) gradients. Both components can drive ATP synthesis, whereas the DeltapH component alone can trigger feedback regulation of the antenna. It has often been suggested that a relatively large pmf is needed to sustain the energetic contributions of the ATP synthase reaction (DeltaG(ATP)), and that the Deltapsi component is dissipated during illumination, leading to an acidic lumen in the light. We suggest that this is incompatible with the stabilities of lumenal components and the observed activation of downregulation. Recent work on the chloroplast ATP synthase suggests that a more moderate pmf can sustain DeltaG(ATP). In addition, in vivo probes suggest that a substantial fraction of pmf can be stored as Deltapsi. Together, these factors should allow sufficient DeltaG(ATP) to maintain lumen pH in a range where lumenal enzyme activities are nearly optimal, and where the level of NPQ is regulated.
... In chloroplasts, ∆pH is usually considered the basic contributor to ∆µ H + [10,[31][32][33]. The energy stored in the form of ∆pH (∆pH~1.0-1.5 or higher, which is equivalent to 60-90 mV) is sufficient to sustain efficient ATP synthesis, providing the stoichiometric ratio H + /ATP ≥ 4-5 (for references, see [34]). ...
... The steady-state values of ∆ψ are usually insignificant (∆ψ < 20 mV). However, under certain experimental conditions, the deposition of ∆ψ to pmf cannot be ignored [10,[31][32][33]. Although the ∆pH difference is usually considered the dominant component of ∆µ H+ , the relative contributions of ∆ψ and ∆pH to the overall value of ∆µ H+ may vary, depending on the nature and lipid composition of the thylakoid membranes. ...
... Bearing in mind the importance of pmf as a free energy intermediate, it is not surprising that a great number of works in the field of membrane bioenergetics have been devoted to the determination of pmf values and numerical parsing of ∆ψ and ∆pH in energytransducing biological systems (for references, see [3,4,[27][28][29][30][31][32][33][34]). In this section, we present a brief overview of the widely used methods for monitoring the ∆ψ and ∆pH components of pmf by using electrometrical, optical, and electron paramagnetic resonance (EPR) methods. ...
A transmembrane difference in the electrochemical potentials of protons (ΔμH+) serves as a free energy intermediate in energy-transducing organelles of the living cell. The contributions of two components of the ΔμH+ (electrical, Δψ, and concentrational, ΔpH) to the overall ΔμH+ value depend on the nature and lipid composition of the energy-coupling membrane. In this review, we briefly consider several of the most common instrumental (electrometric and EPR) methods for numerical estimations of Δψ and ΔpH. In particular, the kinetics of the flash-induced electrometrical measurements of Δψ in bacterial chromatophores, isolated bacterial reaction centers, and Photosystems I and II of the oxygenic photosynthesis, as well as the use of pH-sensitive molecular indicators and kinetic data regarding pH-dependent electron transport in chloroplasts, have been reviewed. Further perspectives on the application of these methods to solve some fundamental and practical problems of membrane bioenergetics are discussed.
... The main constraint for the photosynthetic electron transfer is to adjust its activity to the amount of light received and to the metabolic needs in terms of NADPH and ATP, despite a variable environment (Kramer et al. 2003;Peltier et al. 2010). When light absorption exceeds the capacity of the electron transfer chain, the overreduction of the photosynthetic chain can lead to the photodestruction of both PSII (Nixon et al. 2010) and PSI (Sonoike 2011) (see also Sect. ...
... Collectively, 1 O 2 , superoxide, H 2 O 2 , and the hydroxyl radical are called reactive oxygen species (ROS). The lumenal pH regulates photoprotection of the two photosystems through the de-excitation of PSII under excess light conditions (Kramer et al. 2003;Holt et al. 2004, and see Sect. 4.4) and the "photosynthetic control," which is the modulation of the activity of the cyt b 6 f by the lumenal pH. ...
... Here again, the flexibility of the ATP/NADPH stoichiometry produced by the photosynthetic machinery is modulated by the pmf, which is increased by alternative electrons flows (AEFs) fueling the ATP synthase (see Sect. 3.2). In plants and green algae, the flexibility of photosynthesis is therefore largely dependent on the pmf, which plays the dual role of driving ATP and regulating photoprotection mechanisms through its osmotic component, the ΔpH (Kramer et al. 2003). ...
... The main constraint for the photosynthetic electron transfer is to adjust its activity to the amount of light received and to the metabolic needs in terms of NADPH and ATP, despite a variable environment (Kramer et al. 2003;Peltier et al. 2010). When light absorption exceeds the capacity of the electron transfer chain, the overreduction of the photosynthetic chain can lead to the photodestruction of both PSII (Nixon et al. 2010) and PSI (Sonoike 2011) (see also Sect. ...
... Collectively, 1 O 2 , superoxide, H 2 O 2 , and the hydroxyl radical are called reactive oxygen species (ROS). The lumenal pH regulates photoprotection of the two photosystems through the de-excitation of PSII under excess light conditions (Kramer et al. 2003;Holt et al. 2004, and see Sect. 4.4) and the "photosynthetic control," which is the modulation of the activity of the cyt b 6 f by the lumenal pH. ...
... Here again, the flexibility of the ATP/NADPH stoichiometry produced by the photosynthetic machinery is modulated by the pmf, which is increased by alternative electrons flows (AEFs) fueling the ATP synthase (see Sect. 3.2). In plants and green algae, the flexibility of photosynthesis is therefore largely dependent on the pmf, which plays the dual role of driving ATP and regulating photoprotection mechanisms through its osmotic component, the ΔpH (Kramer et al. 2003). ...
Photosynthesis in diatoms is performed using the same basic modules as cyanobacteria and plants. It can be regulated on multiple levels depending on the environmental cues, allowing diatoms to adjust their photosynthetic light reaction toward optimum while at the same time minimizing photodamage induced by light. In recent years, tremendous progress has been gained in understanding these acclimation processes, revealing several diatom-specific features. In this chapter, we trace several paths through the photosynthetic electron transport chain to optimize photosynthesis. We review how diatoms repair photoinactivated reaction centers and which mechanisms they have to pre-empt photodamage. Finally, photoprotection is set in an ecophysiological context, highlighting differences in photoprotection of diatoms from different habitats.KeywordsAlternative electron flowDiatomsLhcxNPQPhotoinhibitionPhotosynthesisPhotosystem II repairXanthophyll cycle
... Here, the proton-linked redox chemistry at the quinone reduction (Q i ) site of this enzyme results in the uptake of protons from the stroma. The oxidation of PQH 2 at the quinol oxidation (Q o ) site of the b 6 f complex deposits these protons into the lumen (Kramer et al., 2003). ...
... Thus, the H þ /ATP stoichiometry is expected to be 4.67. As the H þ / 2e À (i.e., H þ /NADPH) yield of LEF is fixed at 6, it is apparent that the ATP/NADPH output due to LEF will be 1.28 molecules of ATP generated per molecule of NADPH, assuming a perfectly coupled system (Kramer et al., 2003 ;Cruz et al., 2005). ...
... This so-called 'photosynthetic control' prevents the accumulation of electrons on PSI, preventing the production of superoxide or other reactive oxygen species that can lead to photodamage. The proton gradient supplied by CEF appears to serve a critical PSI-photoprotective function during fluctuating illumination (as may be experienced naturally as light flecks due to e.g., leaf-and cloud movements), as mutant plants and algae deficient in the CEF pathway frequently exhibit sensitivity to these lighting conditions Kramer et al., 2003;Shikanai, 2014). ...
Photosynthesis must balance how much energy is stored in ATP and NADPH to precisely meet the ratio required for biochemical demands. If this balancing does not occur, the system will fail, leading to photodamage. The balancing processes must be extremely robust to contend with the rapid and unpredictable fluctuations in environmental conditions and metabolic demands that occur in nature. The major mechanisms for balancing the ATP/NADPH budget involve activating and regulating alternative electron transfer processes, such as cyclic electron flow around Photosystem I, that contribute to the thylakoid protonmotive force (pmf), augmenting ATP production and photoprotective mechanisms.
... Within the chloroplast thylakoid membrane, light energy is used to drive charge separation in the photosynthetic reaction centers, photosystem I (PSI) and photosystem II (PSII). These photochemical reactions, and the subsequent operation of the Q-cycle within cytochrome b 6 f (cytb 6 f), result in the movement of electrons and protons across the span of the thylakoid membrane bilayer, generating an electrical potential (Dw) and a chemical gradient of protons (DpH) (Kramer et al., 2003;Malone et al., 2021). This electrochemical gradient is known as the proton motive force (pmf) and is utilized by the thylakoid ATP synthase to drive the endergonic synthesis of ATP in the chloroplast stroma (Nelson and Junge, 2015). ...
... The consensus view that the steady-state transthylakoid pmf consists primarily of DpH, built largely on work with isolated chloroplasts, was later challenged by the emergence of the electrochromic shift (ECS) signal as an in vivo probe of the pmf in intact leaves (Kramer and Sacksteder, 1998;Cruz et al., 2001;Kramer et al., 2003). The Dw component induces an ECS in the Soret peak absorption of chlorophylls and carotenoids in the thylakoid membrane (Witt, 1971(Witt, , 1979Vredenberg, 1997;Bailleul et al., 2010). ...
... This results in the formation of a transient absorption peak $515 nm upon illumination of leaves (Witt, 1971). Since a significant proportion of the ECS signal persisted during continuous illumination, Kramer et al. suggested that in vivo, a larger fraction of pmf is stored as Dw than was suggested by the earlier in vitro work (Kramer and Sacksteder, 1998;Cruz et al., 2001;Kramer et al., 2003). Interestingly, they found that the parsing of the pmf between the Dw and DpH, implied by the ECS measurements, was affected by light intensity and CO 2 availability (Kanazawa and Kramer, 2002;Takizawa et al., 2007). ...
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 ∼515 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 non-photochemical 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 between 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 seconds of continuous illumination under both low and high light conditions. Further implications of these findings for the regulation of photosynthesis are discussed.
... This ΔpH contributes to the formation of proton motive force (pmf) in addition to the membrane potential formed across the thylakoid membrane (Δc) that results from the uneven distribution of ions across the membrane. The pmf energizes ATP synthesis via F o F 1 -ATP synthase in chloroplasts (Kramer et al., 2003;Soga et al., 2017) and thus influences the efficiency of the light reactions. ...
... Both ΔpH and Δc contribute to pmf, but only ΔpH down-regulates electron transport. To optimize the operation of the accelerator (ATP synthesis) and the brake on electron transport, it is necessary to precisely regulate the ratio of the two pmf components as well as the total size of pmf (Cruz et al., 2001;Kramer et al., 2003). Several channels and antiporters localized to the thylakoid membrane regulate the partitioning of the pmf components (Spetea et al., 2017). ...
... The PGR5/PGRL1-dependent CET is essential to induce ΔpH-dependent regulation: the energy-dependent quenching, monitored by NPQ, and the donor-side regulation of PSI at the Cyt b 6 f complex monitored by Y(ND) (Munekage et al., 2004;Suorsa et al., 2012). On the other hand, channels and transporters localized to the thylakoid membrane regulate the partitioning of pmf components (Kramer et al., 2003, Spetea et al., 2017. The putative H 1 /K 1 antiporter KEA3 was discovered only recently, and little is known about it, especially regarding the regulation of its activity (Armbruster et al., 2014;Kunz et al., 2014;Wang et al., 2017). ...
In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around photosystem I (CET). K+ efflux antiporter 3 (KEA3) is a putative thylakoid H+/K+ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction 2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis thaliana. The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m-2 s-1 after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent downregulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 (PGR5)-overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m-2 s-1, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent downregulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO2 fixation was delayed after overnight dark adaptation.
... Generation of ΔрН is accompanied by a decrease in the lumen pH from 7.0 (in the dark) to pH 5.7-5.8 [49,50]. When pH in the lumen is above 7.0, OEC in some PSII preparations is irreversibly inhibited [9]. ...
... Agreement of acidity at which protection of PSII from photoinhibition is elevated (pH 5.7) with pH value arising in the lumen upon generation of transmembrane pH gradient implies that pH-dependent structural transition at pH 5.7 may play the role of a novel (earlier unknown) pH-dependent mechanism ensuring self-defense of photosynthetic apparatus against photoinactivation. A diagram of such a mechanism shown in Fig. 4 envisages the following: in thylakoid membranes, illumination of PSII is accompanied by generation of ΔpH on the thylakoid membrane and a decrease in pH in the lumen to the value of ≈5.7 [49,50], i.e., to pH ensuring the strongest defense against photoinhibition. Thus, the effect we discovered accounts for an additional mechanism of PSII defense against photoinhibition in the light. ...
Photosystem II (PSII) of the photosynthetic apparatus in oxygenic organisms contains a catalytic center that performs one of the most important reactions in bioenergetics: light-dependent water oxidation to molecular oxygen. The catalytic center is a Mn 4 CaO 5 cluster consisting of four cations of manganese and one calcium cation linked by oxygen bridges. The authors reported earlier that a structural transition occurs at pH 5.7 in the cluster resulting in changes in manganese cation(s) redox potential and elevation of the Mn‑clus-ter resistance to reducing agents. The discovered effect was examined in a series of investigations that are reviewed in this work. It was found that, at pH 5.7, Fe(II) cations replace not two manganese cations as it happens at pH 6.5 but only one cation; as a result, a chimeric Mn 3 Fe 1 cluster is produced. In the presence of exogenous calcium ions, membrane preparations of PSII with such a chimeric cluster are capable of evolving oxygen in the light (at a rate of approximately 25% of the rate in native PSII). It was found that photoinhibition that greatly depends on the processes of oxidation or reduction at pH 5.7 slows down as compared with pH 6.5. PSII preparations were also more resistant to thermal inactivation at pH 5.7 than at pH 6.5. However, in PSII preparations lacking manganese cations in the oxygen-evolving complex, the rates of photoinhibition at pH 6.5 and 5.7 did not differ. In thylakoid membranes, protonophores that abolish the proton gradient and increase pH in the lumen (where the manganese cluster is located) from 5.7 to 7.0 considerably elevated the rate of PSII photoinhibition. It is assumed that the structural transition in the Mn-cluster at pH 5.7 is involved in the mechanisms of PSII defense against photoinhibition.
... The F o F 1 -ATP synthase in chloroplasts and cyanobacteria produces adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate. This reaction is driven by a proton electrochemical gradient (ΔμH + ) across the thylakoid membranes formed by the photosynthetic electron transfer system (1,2). This enzyme is a large complex (>500 kDa) consisting of a membrane-embedded F 0 component (abb'c x ; x is the variable number among spices) that functions as a proton channel and a membrane-peripheral F 1 component (α 3 β 3 γδε) that contains three catalytic sites. ...
... The ATP synthesis/ hydrolysis reactions are catalyzed by the cooperation of these two parts (3). Under light conditions, the ΔμH + formed by the photosynthetic electron transfer system induces rotation of the central stalk (γεc x ) of F o F 1 , and ATP is generated (1,4). In contrast, under dark conditions, several inhibitory mechanisms, such as Mg-bound ADP (MgADP) inhibition and ε inhibition, inhibit enzyme activity and prevent the occurrence of a wasteful ATP hydrolysis reaction (5)(6)(7)(8)(9). ...
Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the β-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the β-hairpin and the redox loop domains specific to CFoCF1.
... Non-photochemical quenching (NPQ), a composite of PSII-associated, photoprotective processes (Müller et al., 2001), functions to benignly dissipate excess absorbed light energy, thereby minimizing the potential for photooxidative damage (Niyogi, 1999). The predominant component of NPQ harmlessly dissipates excess energy as heat and is referred to as energy-dependent quenching of excitons (q E ; Crofts & Yerkes, 1994), the rapid reversibility of which, as during the transient shading of a leaf during the passing of a cloud or wind-driven shading by the chaotic movements of leaves within canopies, is partially dependent on processes that reversibly modulate the magnitude of the ΔpH component of the pmf (Bennett et al., 2019;Kramer et al., 2003). ...
... CEF has been proposed to have various roles, including protection of the photosynthetic apparatus via modulation of q E Avenson et al., 2005;Kanazawa & Kramer, 2002), thereby minimizing the production of ROS (Foyer et al., 2012). Since CEF acidifies the thylakoid lumen over and above that which is associated with LEF, it has also been proposed to play a role in balancing the output ratio of ATP:NADPH with fluctuating metabolic demands (Kramer et al., 2003;Livingston et al., 2010). ...
Abstract Pulsed amplitude modulation (PAM) chlorophyll a fluorescence provides information about photosynthetic energy transduction. When reliably measured, chlorophyll a fluorescence provides detailed information about critical in vivo photosynthetic processes. Such information has recently provided novel and critical insights into how the yield potential of crops can be improved and it is being used to understand remotely sensed fluorescence, which is termed solar‐induced fluorescence and will be solely measured by a satellite scheduled to be launched this year. While PAM chlorophyll a fluorometers measure fluorescence intensity per se, herein we articulate the axiomatic criteria by which instrumentally detected intensities can be assumed to assess fluorescence yield, a phenomenon quite different than fluorescence intensity and one that provides critical insight about how solar energy is variably partitioned into the biosphere. An integrated mathematical, phenomenological, and practical discussion of many useful chlorophyll a fluorescence parameters is presented. We draw attention to, and provide examples of, potential uncertainties that can result from incorrect methodological practices and potentially problematic instrumental design features. Fundamentals of fluorescence measurements are discussed, including the major assumptions underlying the signals and the methodological caveats about taking measurements during both dark‐ and light‐adapted conditions. Key fluorescence parameters are discussed in the context of recent applications under environmental stress. Nuanced information that can be gleaned from intra‐comparisons of fluorescence‐derived parameters and intercomparisons of fluorescence‐derived parameters with those based on other techniques is elucidated.
... Parmi les mécanismes permettant de protéger la chaîne photosynthétique, la dissipation non photochimique de l'excitation (qE) dans le PSII est régulée par le ΔpH. En effet, ce phénomène est fortement lié à la conversion de pigments, le cycle des xanthophylles présenté plus haut, qui est dépendant du pH luménal, et de sous-unités du PSII dont la protonation, en conditions de lumen acide, participe au mécanisme de dissipation (Kramer et al, 2003). Ce phénomène est visualisé par une diminution de la fluorescence de la chlorophylle et porte parfois par abus de langage le nom de NPQ (pour « non-photochemical quenching », voir section suivante et Méthodes). ...
... Comme dans la lignée verte (Peltier et al., 2010 ;Kramer et al., 2003), l'ensemble de ces transferts d'électrons alternatifs, a un double rôle : ils sont importants pour la photoprotection des deux photosystèmes en modulant la composante osmotique de la pmf, et ils jouent un rôle primordial dans l'ajustement du rapport ATP/ NADPH. Il est raisonnable de penser que ce rôle de régulateur de la photosynthèse est particulièrement crucial lorsque de changements rapides de la disponibilité en CO2, de l'intensité lumineuse et/ou quand la demande métabolique varie. ...
Une des principales forces qui façonnent la dynamique et la structure des communautés phytoplanctoniques est l'inhibition directe du métabolisme des concurrents grâce à l'utilisation de métabolites secondaires. Mais notre compréhension de ce phénomène, appelé allélopathie, reste limitée. La photosynthèse est une sonde idéale pour étudier les interactions allélopathiques entre microalgues marines, car elle en est une des cibles principales. Nous avons validé une approche nouvelle, permettant de disséquer les activités photosynthétiques de chaque microalgue au sein d’un mélange, permettant de dévoiler des interactions allélopathiques ciblant la photosynthèse. Cette thèse se concentre sur le plaste des diatomées, qui, au-delà d’être le centre énergétique de la cellule, s’avèrent aussi être la cible des composés allélochimiques libérés par leurs compétiteurs. Cette approche méthodologique a permis de montrer l’inhibition de l’activité photosynthétique de la diatomée T. pseudonana par des composés allélochimiques libérés par le dinoflagellé A. carterae. Cette inhibition implique la dissipation du gradient électrochimique de proton généré à travers les thylacoïdes, probablement par des molécules de type amphidinols formant des pores sur les membranes biologiques. Au cours de deux collaborations, nous avons montré deux autres interactions allélopathiques ciblant le plaste des diatomées : le rôle des quinolones libérées par des bactéries, sur les chaînes de transport d’électron photosynthétique et respiratoire de la diatomée P. tricornutum, les effets du filtrat du dinoflagellé A. minutum sur les membranes cytoplasmiques et chloroplastiques de la diatomée C. muelleri.
... However, few studies assessed the effect of pH or CO 2 -induced acidification on microalgae antioxidant activity (Guedes et al., 2011c;Xia et al., 2018). Yet, pH and CO 2 -induced acidification affects dissolved inorganic carbon availability, intracellular acid base balance, structural rearrangement of pigment systems, and therefore, may influence growth, carbon assimilation, energy demand to maintain the membrane electrochemical potential and enzyme activity, and intracellular oxidative stress (Goss and Garab, 2001;Kramer et al., 2003;Milligan et al., 2009;Xia et al., 2018). Changes in carbon fixation will also modify the cellular concentration of ATP and NADPH, which in turn may modify photochemical processes and energy dissipation pathways (Takahashi and Murata, 2005). ...
... Low internal pH is known to provoke inhibition of Rubisco which reduces consumption of NADPH and ATP and decrease electron sink from the photosynthetic electron transport chain (Miyachi et al., 2003;Ptushenko and Solovchenko, 2016). In addition, NPQ (xanthophyll cycle and protonation of PsbS protein) is known to be triggered by acidification of the thylakoid lumen when chloroplasts are illuminated (Goss and Garab, 2001;Kramer et al., 2003). It could explain the negative impact of CO 2 acidification on photophysiological parameters. ...
Tetraselmis sp. was selected for its antioxidant activity owing to its high lipid peroxidation inhibition capacity. With the aim to monitor culture conditions to improve antioxidant activity, effects of CO2-induced acidification on Tetraselmis growth, elemental composition, photosynthetic parameters and antioxidant activity were determined. Two pH values were tested (6.5 and 8.5) in batch and continuous cultures in photobioreactors. Acidification enhanced cell growth under both culture methods. However, the microalgae physiological state was healthier at pH 8.5 than at pH 6.5. Indeed, photosynthetic parameters measured with pulse amplitude modulated (PAM) fluorometry showed a decrease in the photosystem II (PSII) efficiency at pH 6.5 in batch culture. Yet, with the exception of the PSII recovering capacity, photosynthetic parameters were similar in continuous culture at both pH. These results suggest that lowering pH through CO2-induced acidification may induce a lower conversion of light to chemical energy especially when coupled with N-limitation and/or under un-balanced culture conditions. The highest antioxidant activity was measured in continuous culture at pH 6.5 with an IC50 of 3.44 ± 0.6 µg mL-1, which is close to the IC50 of reference compounds (trolox and α-tocopherol). In addition, the principal component analysis revealed a strong link between the antioxidant activity and the culture method, the photophysiological state and the nitrogen cell quota and C:N ratio of Tetraselmis sp.. These results highlight Tetraselmis sp. as a species of interest for natural antioxidant production and the potential of PAM fluorometry to monitor culture for production of biomass with a high antioxidant activity.
... Indeed, when these contributions were removed pmf was almost entirely partitioned into ΔpH (Wilson et al. 2021), consistent with experiments on intact chloroplasts (Vredenberg 1997). Nonetheless, even when the ECS signal is partitioned into nominal ΔpH as per Kramer et al. (2003), still no correlation is observed with Y(ND) in leaves (Fig. 4G). Collectively, these data show that no simple relationship exists in intact leaves between KP700, Y(ND) and pmf. ...
Photosynthetic control (PCON) is a protective mechanism that prevents light-induced damage to photosystem I (PSI) by ensuring the rate of NADPH and ATP production via linear electron transfer (LET) is balanced by their consumption in the CO2 fixation reactions. Protection of PSI is a priority for plants since they lack a dedicated rapid-repair cycle for this complex, meaning that any damage leads to prolonged photoinhibition and decreased growth. The imbalance between LET and the CO2 fixation reactions is sensed at the level of the transthylakoid ΔpH, which increases when light is in excess. The canonical mechanism of PCON involves feedback control by ΔpH on the plastoquinol oxidation step of LET at cytochrome b6f. PCON thereby maintains the PSI special pair chlorophylls (P700) in an oxidized state, that allows excess electrons unused in the CO2 fixation reactions to be safely quenched via charge recombination. In this review we focus on angiosperms, considering how photo-oxidative damage to PSI comes about, explore the consequences of PSI photoinhibition on photosynthesis and growth, discuss recent progress in understanding PCON regulation, and finally consider the prospects for its future manipulation in crop plants to improve photosynthetic efficiency.
... Upon illumination, the electron and proton transport reaction deposits protons into the lumen, generating a pmf, which is composed of two components: the electric field (Δψ) and the proton gradient (ΔpH). Both pmf components drive the synthesis of ATP at the chloroplast ATP synthase (Kramer et al., 2003). The initial form of pmf is exclusively stored as the electric field component due to the lower capacity of electric capacitance and higher buffering capacity of ΔpH in the lumen (Cruz et al., 2005;Kanazawa & Kramer, 2002;Takizawa et al., 2008). ...
Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force ( pmf ) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.
... ECS estimates light-dependent photosynthesis-driven transthylakoid proton flux using IDEA spectrophotometer (Baker et al. 2007) (Fig. 2). We used dark interval relaxation kinetics (DIRK) to observe the shifts in the membrane potential (electrochromic signal at 520 nm) (Kramer et al. 2003). The proton motive force (pmf) was estimated from the total ECS signal (ECS t ) in C. reinhardtii under both low and high salinity conditions (Fig. 2a). ...
While PSI-driven cyclic electron flow (CEF) and assembly of thylakoid supercomplexes have been described in model organisms like Chlamydomonas reinhardtii, open questions remain regarding their contributions to survival under long-term stress. The Antarctic halophyte, C. priscuii UWO241 (UWO241), possesses constitutive high CEF rates and a stable PSI-supercomplex as a consequence of adaptation to permanent low temperatures and high salinity. To understand whether CEF represents a broader acclimation strategy to short- and long-term stress, we compared high salt acclimation between the halotolerant UWO241, the salt-sensitive model, C. reinhardtii, and a moderately halotolerant Antarctic green alga, C. sp. ICE-MDV (ICE-MDV). CEF was activated under high salt and associated with increased non-photochemical quenching in all three Chlamydomonas species. Furthermore, high salt-acclimated cells of either strain formed a PSI-supercomplex, while state transition capacity was attenuated. How the CEF-associated PSI-supercomplex interferes with state transition response is not yet known. We present a model for interaction between PSI-supercomplex formation, state transitions, and the important role of CEF for survival during long-term exposure to high salt.
... following a second turnover at the Q o -site. Consequently, these electron transport reactions are coupled to proton transfer from the electrochemically negative to the positive side across the membrane, contributing to the production of electrochemical proton gradient, or proton motive force (pmf), which is essential for a variety of cellular activities such as ATP synthesis (Kramer et al., 2003). ...
In photosynthetic green sulfur bacteria, the electron transfer reaction from menaquinol:cytochrome c oxidoreductase to the P840 reaction center (RC) complex occurs directly without any involvement of soluble electron carrier protein(s). X-ray crystallography has determined the three-dimensional structures of the soluble domains of the CT0073 gene product and Rieske iron-sulfur protein (ISP). The former is a mono-heme cytochrome c with an α-absorption peak at 556 nm. The overall fold of the soluble domain of cytochrome c-556 (designated as cyt c-556sol) consists of four α-helices and is very similar to that of water-soluble cyt c-554 that independently functions as an electron donor to the P840 RC complex. However, the latter's remarkably long and flexible loop between the α3 and α4 helices seems to make it impossible to be a substitute for the former. The structure of the soluble domain of the Rieske ISP (Rieskesol protein) shows a typical β-sheets-dominated fold with a small cluster-binding and a large subdomain. The architecture of the Rieskesol protein is bilobal and belongs to those of b6f-type Rieske ISPs. Nuclear magnetic resonance (NMR) measurements revealed weak non-polar but specific interaction sites on Rieskesol protein when mixed with cyt c-556sol. Therefore, menaquinol:cytochrome c oxidoreductase in green sulfur bacteria features a Rieske/cytb complex tightly associated with membrane-anchored cyt c-556.
... The results show that direct injury to PSI by NSAID drugs is minimal and that photochemical reactions connected with PSII and the Hill reaction in the oxygen-evolving centre on the donor side and electron transport on the acceptor side of PSII are probably the main targets of these drugs in primary photosynthetic processes. Limitations of both these processes could result in a reduced pool of H + ions in the thylakoid lumen, resulting in a limited production of ATP (Kramer et al., 2003). ...
Non-steroidal anti-inflammatory drugs (NSAID) are recently monitored in the aquatic environment. Naproxen (NPX), paracetamol (PCT) and their transformation products can influence the biochemical and physiological processes at the sub-cellular and cellular levels taking part in the growth and development of plants. This study aimed to compare the effects of NPX and PCT, drugs with different physico-chemical properties, on the growth and photosynthetic processes in Lemna minor during a short-term (7 days) exposure. Although duckweed took up more than five times higher amount of PCT as compared to NPX (275.88 µg/g dry weight to 43.22 µg/g when treated with 10 mg/L), only NPX limited the number of new plants by 9% and 26% under 1 and 10 mg/L, respectively, and increased their dry weight (by 18% under 10 mg/L) and leaf area per plant. A considerable (by 30%) drop in the content of photosynthetic pigments under 10 mg/L treatment by both drugs did not significantly affect the efficiency of the primary processes of photosynthesis. Values of induced chlorophyll fluorescence parameters (F0, FV/FM, ΦII, and NPQ) showed just a mild stimulation by PCT and a negative effect by NPX (by up to 10%), especially on the function of photosystem II and electron transport in both intact duckweed plants and isolated chloroplasts. Lowered efficiency of Hill reaction activity (by more than 10% under 0.1 - 10 mg/L treatments) in isolated chloroplasts suspension proved the only inhibition effect of PCT to primary photosynthetic processes. In intact plants, higher treatments (0.5 - 10 mg/L) by both NPX and PCT induced an increase in RuBisCO content. The results prove that the potential effect of various drugs on plants is hard to generalise.
... Across the membrane, the difference in charge imposed by this proton gradient comprises a membrane potential (Dy) and the difference in pH generates a pH gradient (DpH) (Cruz et al., 2001). Together, these comprise the protonmotive force (Dp) for ATP synthesis via the ATP synthase (Nicholls and Ferguson, 2002;Kramer et al., 2003). The chloroplast ATP synthase is comprised of two complexes, the CF o in the membrane, and the CF 1 in the stroma (Hahn et al., 2018). ...
Humans have been harnessing biology to make valuable compounds for generations. From beer and biofuels to pharmaceuticals, biology provides an efficient alternative to industrial processes. With the continuing advancement of molecular tools to genetically modify organisms, biotechnology is poised to solve urgent global problems related to environment, increasing population, and public health. However, the light dependent reactions of photosynthesis are constrained to produce a fixed stoichiometry of ATP and reducing equivalents that may not match the newly introduced synthetic metabolism, leading to inefficiency or damage. While photosynthetic organisms have evolved several ways to modify the ATP/NADPH output from their thylakoid electron transport chain, it is unknown if the native energy balancing mechanisms grant enough flexibility to match the demands of the synthetic metabolism. In this review we discuss the role of photosynthesis in the biotech industry, and the energetic considerations of using photosynthesis to power synthetic biology.
... Specifically, a high fraction of pmf stored as the ΔΨ component is physiologically significant during transitions from low to high light (Wang and Shikanai, 2019;Basso et al., 2020;Correa Galvis et al., 2020;Lazár et al., 2022). Nevertheless, the effect of ΔpH on regulating photosynthesis has always been a hot topic (Li et al., 2021;Kramer et al., 1999Kramer et al., , 2003Avenson et al., 2005;Zaks et al., 2012) but less endeavor has been made on the evaluation of the actual transthylakoid potential difference (ΔΨ) from a theoretical perspective, which likely reflects the computational complexity to determine ΔΨ when all different types of ions in situ together with the binding of cations to the TM-surface are both taken into account. However, some works modeled ΔΨ using different approaches and complexity of the models (Van Kooten et al., 1986;Riznichenko et al., 1999;Lebedeva et al., 2002;Beliaeva et al., 2011;Belyaeva et al., 2016Belyaeva et al., , 2019Zhu et al., 2013). ...
The light-induced transthylakoid membrane potential (ΔΨ) can not only drive the ATP synthesis through the ATP-synthase in chloroplasts but serve as an essential modifier in the acclimation of photosynthesis to fluctuating light conditions. It has been manifested that during photosynthesis, the light-induced ΔΨ is responsive to multiple factors among which the ion channels/transporters (e.g., V–K⁺, VCCN1, and KEA3) are key to adjust the ion distribution on the two sides of the thylakoid membrane and hence shape the kinetics of ΔΨ. However, an in-depth mechanistic understanding of ion fluxes on adjusting the transthylakoid electric potentials is still unclear. This lack of a mechanistic understanding is due to the experimental difficulty of closely observing ion fluxes in vivo and also hacking the evolution of parameters in a highly intertwined photosynthetic network. In this work, a computer model was applied to investigate the roles of ion fluxes on adjusting transthylakoid electric potentials upon a temporal cycle of a period of high illumination followed by a dark-adapted phase. The computing data revealed that, firstly, upon illumination, the dissipation of the steady-ΔΨ by ∼10 mV is contributed from the V–K⁺-driven K⁺ flux whilst ∼8 mV of the steady-ΔΨ is dissipated by the VCCN1-pumped Cl⁻ flux, but there were no appreciable KEA3-evoked variations on ΔΨ; secondly, on transition from high light to darkness, V–K⁺ and KEA3 are serving as major contributors whereas VCCN1 taking a counterbalancing part in shaping a standard trace of ECS (electrochromic shift) which commonly shows a sharp fall to a minimum before returning to the baseline in darkness. Besides, the functional consequences on components of ΔΨ adjusted by every particular ion channel/transporter were also explored. By employing the model, we bring evidence that particular thylakoid-harbored proteins, namely V–K⁺, VCCN1, and KEA3, function by distinct mechanisms in the dynamic adjustment of electric potentials.
... Light-induced photosynthetic electron transfer, coupled with proton translocation, generates ΔΨ and ΔpH across thylakoid membranes (Wilson et al., 2021). The formation of ΔpH and reduction in luminal pH are essential for photosynthetic regulatory mechanisms including the activation of NPQ (Schaller et al., 2014;Chmeliov et al., 2016Chmeliov et al., , 2019Ruban, 2016) and photosynthetic control of cytochrome b 6 f (Cytb 6 f) activity (Hope et al., 1994;Kramer et al., 2003). Moreover, ΔpH and ΔΨ are thermodynamically equivalent components of pmf (Figure 2), following Mitchell's chemiosmotic theory (Mitchell, 1961(Mitchell, , 2011Hangarter and Good, 1982;Wilson et al., 2021), which is indicated in the following equation: ...
The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.
... The pmf across the thylakoid membranes comprise a proton gradient (∆pH) and a membrane potential (∆Ψ), both of them can drive ATP synthesis via the chloroplast ATP synthase [19][20][21]. In addition, ∆pH is a key signal for photosynthetic regulation [15,22,23]. ...
Generally, regulation of cyclic electron flow (CEF) and chloroplast ATP synthase play key roles in photoprotection for photosystems I and II (PSI and PSII) in C3 and C4 plants, especially when CO2 assimilation is restricted. However, how CAM plants protect PSI and PSII when CO2 assimilation is restricted is largely known. In the present study, we measured PSI, PSII, and electrochromic shift signals in the CAM plant Vanilla planifolia. The quantum yields of PSI and PSII photochemistry largely decreased in the afternoon compared to in the morning, indicating that CO2 assimilation was strongly restricted in the afternoon. Meanwhile, non-photochemical quenching (NPQ) in PSII and the donor side limitation of PSI (Y(ND)) significantly increased to protect PSI and PSII. Under such conditions, proton gradient (∆pH) across the thylakoid membranes largely increased and CEF was slightly stimulated, indicating that the increased ∆pH was not caused by the regulation of CEF. In contrast, the activity of chloroplast ATP synthase (gH+) largely decreased in the afternoon. At a given proton flux, the decreasing gH+ increased ∆pH and thus contributed to the enhancement of NPQ and Y(ND). Therefore, in the CAM plant V. planifolia, the ∆pH-dependent photoprotective mechanism is mainly regulated by the regulation of gH+ rather than CEF when CO2 assimilation is restricted.
... To investigate the reasons behind the differences in NPQ between the slugs and the algae, the ECS signal during a strong light pulse was measured from ambient air grown samples of both organisms (Fig. 4A in Paper I). The short ECS measurements did not allow to break the signal down to its two individual components (electric field and ΔpH) and the ECS signal during the light pulse was taken as an indicator of the total pmf in the thylakoid membranes (Kramer et al. 2003). The pmf kept on building up during most of the strong light pulse in the slugs, whereas the pmf in A. acetabulum dissipated to a stable level after the initial spike in thylakoid membrane energization caused by the sudden light burst. ...
Certain sea slugs “steal” the photosynthetic cellular organelles, the plastids, from their prey algae and incorporate them, still functional, inside their own cells. These animals can then remain photosynthetic for months. The redox reactions of photosynthesis are associated with inevitable damage that needs to be constantly repaired. Running photosynthesis with plastids isolated from their algal cell should not be possible, as the algal nucleus that encodes essential maintenance proteins of the photosynthetic machinery is absent in the slug cells. How do photosynthetic sea slugs then avoid or repair the oxidative damage that their plastids should be facing? In my thesis, I have tackled this question by comparing the differences in photosynthetic electron transfer between the photosynthetic sea slug Elysia timida and the source of its plastids, the alga Acetabularia acetabulum. In addition, I compared the rates of photodamage to the plastids in the slugs and in their prey algae. I used the alga Vaucheria litorea, the prey of the slug Elysia chlorotica, to investigate the intrinsic properties of V. litorea plastids that could help explain how these plastids tolerate isolation.
... The absolute pH dependency of quenching in P. tricornutum (apparent pKa of 4.7, Fig. 1c) was shifted towards more acidic values than in green algae (apparent pKa of 6.2 in Chlamydomonas reinhardtii; Tian et al., 2019). Thus, quenching could reflect photosystem II (PSII) photodamage, which is expected at such low lumen pH values (Krieger & Weis, 1993;Spetea et al., 1997;Kramer et al., 2003). However, we found a very similar relationship between NPQ and PSII photochemical quantum yield (F v /F m ) during a HL to low light (LL) relaxation and the acid to neutral pH transition (Fig. S2). ...
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.
... Notably, levels of β-carotene (β-Car), which associates with PSII core proteins, did not vary significantly among the different genotypes. In addition, in both mutants, the pale green phenotype was associated with an increase in photosynthetic performance, as revealed by the higher Φ II values and the increased oxidation levels of the plastoquinone (PQ) pooli.e., increased qL values (Kramer et al., 2003;Henikoff et al., 2004;Tadini et al., 2012) as measured with the Imaging-PAM fluorometer (Fig. 5A). ...
Truncated antenna size of photosystems and lower leaf chlorophyll content has been shown to increase photosynthetic efficiency and biomass accumulation in microalgae, cyanobacteria and higher plants grown under high-density cultivation conditions. Here, we have asked whether this strategy is also applicable to a major crop by characterising the barley mutant happy under the sun 1 (hus1). The pale green phenotype of hus1 is due to a 50% reduction in the chlorophyll content of leaves, owing to a premature stop codon in the HvcpSRP43 gene for the 43-kDa chloroplast Signal Recognition Particle (cpSRP43). The HvcpSRP43 protein is responsible for the uploading of photosystem antenna proteins into the thylakoid membranes, and its truncation results in a smaller photosystem antenna size. Besides a detailed molecular and physiological characterization of the mutant grown under controlled greenhouse conditions, we show that the agronomic performance of hus1 plants, in terms of total biomass production and grain yield under standard field conditions, is comparable to that of control plants. The results are discussed in terms of the potential benefits of the hus1 phenotype, and of natural allelic variants of the HvcpSRP43 locus, with respect to productivity and mitigation of climate change.
... This represents the driving force of ATP (adenosine triphosphate) synthesis via chemiosmotic coupling. [6][7][8] ATP constitutes the central energy storage molecule within all living systems and is thus a crucial cofactor for many energy-dependent biochemical reactions, such as CO 2 fixation reactions in the Calvin-Benson-Bassham cycle. [9] Using visible light as a switchable stimulus or energy source, several strategies for the semi-artificial photoinduced production of ATP have been reported. ...
During the light‐dependent reaction of photosynthesis, green plants couple photoinduced cascades of redox reactions with transmembrane proton translocations to generate reducing equivalents and chemical energy in the form of NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate), respectively. We mimic these basic processes by combining molecular ruthenium polypyridine‐based photocatalysts and inverted vesicles derived from Escherichia coli. Upon irradiation with visible light, the interplay of photocatalytic nicotinamide reduction and enzymatic membrane‐located respiration leads to the simultaneous formation of two biologically active cofactors, NADH (nicotinamide adenine dinucleotide) and ATP, respectively. This inorganic‐biologic hybrid system thus emulates the cofactor delivering function of an active chloroplast.
... Alternatively, lumen acidification can also be associated with an increase in the fraction of pmf that is stored as ΔpH, by controlling the flow of counterions across the thylakoid membrane, altering the partitioning of pmf in ΔpH and Δψ [10,16,56]. In this case, acidification may occur with little or no increases in total pmf, or the rates of proton influx [57], though the current field-based data do not allow us to directly distinguish these possibilities. royalsocietypublishing.org/journal/rsos R. Soc. ...
The responses of plant photosynthesis to rapid fluctuations in environmental conditions are critical for efficient conversion of light energy. These responses are not well-seen laboratory conditions and are difficult to probe in field environments. We demonstrate an open science approach to this problem that combines multifaceted measurements of photosynthesis and environmental conditions, and an unsupervised statistical clustering approach. In a selected set of data on mint ( Mentha sp.), we show that ‘light potentials’ for linear electron flow and non-photochemical quenching (NPQ) upon rapid light increases are strongly suppressed in leaves previously exposed to low ambient photosynthetically active radiation (PAR) or low leaf temperatures, factors that can act both independently and cooperatively. Further analyses allowed us to test specific mechanisms. With decreasing leaf temperature or PAR, limitations to photosynthesis during high light fluctuations shifted from rapidly induced NPQ to photosynthetic control of electron flow at the cytochrome b 6 f complex. At low temperatures, high light induced lumen acidification, but did not induce NPQ, leading to accumulation of reduced electron transfer intermediates, probably inducing photodamage, revealing a potential target for improving the efficiency and robustness of photosynthesis. We discuss the implications of the approach for open science efforts to understand and improve crop productivity.
... The linear electron transport from water to NADP + is driven by a series of photochemical reactions catalyzed by PSII and PSI, coupled with proton translocation across the thylakoid membrane to generate proton motive force. The proton motive force consists of the proton concentration gradient (DpH) and membrane potential (DW) and is utilized in ATP synthesis (Kramer et al., 2003). However, this linear electron transport does not satisfy the ATP/NADPH production ratio of 1.5-1.67, ...
Plastid terminal oxidase (PTOX) accepts electrons from plastoquinol to reduce molecular oxygen to water. We introduced the gene encoding Chlamydomonas reinhardtii (Cr)PTOX2 into the Arabidopsis (Arabidopsis thaliana) wild type (WT) and proton gradient regulation5 (pgr5) mutant defective in cyclic electron transport around photosystem I (PSI). Accumulation of CrPTOX2 only mildly affected photosynthetic electron transport in the WT background during steady-state photosynthesis but partly complemented the induction of nonphotochemical quenching (NPQ) in the pgr5 background. During the induction of photosynthesis by actinic light (AL) of 130 µmol photons m−2 s−1, the high level of PSII yield (Y(II)) was induced immediately after the onset of AL in WT plants accumulating CrPTOX2. NPQ was more rapidly induced in the transgenic plants than in WT plants. P700 was also oxidized immediately after the onset of AL. Although CrPTOX2 does not directly induce a proton concentration gradient (ΔpH) across the thylakoid membrane, the coupled reaction of PSII generated ΔpH to induce NPQ and the downregulation of the cytochrome b6f complex. Rapid induction of Y(II) and NPQ was also observed in the pgr5 plants accumulating CrPTOX2. In contrast to the WT background, P700 was not oxidized in the pgr5 background. Although the thylakoid lumen was acidified by CrPTOX2, PGR5 was essential for oxidizing P700. In addition to acidification of the thylakoid lumen to downregulate the cytochrome b6f complex (donor-side regulation), PGR5 may be required for draining electrons from PSI by transferring them to the plastoquinone pool. We propose a reevaluation of the contribution of this acceptor-side regulation by PGR5 in the photoprotection of PSI.
... This sets up a proton gradient pH across thylakoid membrane. pH and ψ, the thylakoid membrane potential, together constitute the pmf, which drives stromal ATP production via ATP synthase (Kramer et al., 2003). NADPH and ATP fuel the Calvin Bassham Benson Cycle (CBBC) resulting in CO 2 -fixation, synthesis of triose-phosphates, and regeneration of NADP (Scheibe, 2004;Kramer and Evans, 2011;Dietz et al., 2016). ...
Plant productivity greatly relies on a flawless concerted function of the two photosystems (PS) in the chloroplast thylakoid membrane. While damage to PSII can be rapidly resolved, PSI repair is complex and time-consuming. A major threat to PSI integrity is acceptor side limitation e.g., through a lack of stromal NADP ready to accept electrons from PSI. This situation can occur when oscillations in growth light and temperature result in a drop of CO2 fixation and concomitant NADPH consumption. Plants have evolved a plethora of pathways at the thylakoid membrane but also in the chloroplast stroma to avoid acceptor side limitation. For instance, reduced ferredoxin can be recycled in cyclic electron flow or reducing equivalents can be indirectly exported from the organelle via the malate valve, a coordinated effort of stromal malate dehydrogenases and envelope membrane transporters. For a long time, the NADP(H) was assumed to be the only nicotinamide adenine dinucleotide coenzyme to participate in diurnal chloroplast metabolism and the export of reductants via this route. However, over the last years several independent studies have indicated an underappreciated role for NAD(H) in illuminated leaf plastids. In part, it explains the existence of the light-independent NAD-specific malate dehydrogenase in the stroma. We review the history of the malate valve and discuss the potential role of stromal NAD(H) for the plant survival under adverse growth conditions as well as the option to utilize the stromal NAD(H) pool to mitigate PSI damage.
... The ECS signal was measured by the change in absorbance of thylakoid pigments at 520 nm during the application of light-dark interval kinetics (Baker et al., 2007). The total amplitude of ECS signal (ECS t ) was used to estimate the total proton motive force (pmf) across thylakoid membranes (Kramer et al., 2003). UWO 241 grown in HS exhibited 6-to 7.5-fold higher ECS t than that of LS-grown cells under all light intensities ( Fig. 2A), suggesting that HS-grown cells generate higher pmf than LS-grown cells at the same light intensity. ...
Under environmental stress, plants and algae employ a variety of strategies to protect the photosynthetic apparatus and maintain photostasis. To date, most studies on stress acclimation have focused on model organisms which possess limited to no tolerance to stressful extremes. We studied the ability of the Antarctic alga Chlamydomonas sp. UWO 241 (UWO 241) to acclimate to low temperature, high salinity or high light. UWO 241 maintained robust growth and photosynthetic activity at levels of temperature (2 °C) and salinity (700 mM NaCl) which were nonpermissive for a mesophilic sister species, Chlamydomonas raudensis SAG 49.72 (SAG 49.72). Acclimation in the mesophile involved classic mechanisms, including downregulation of light harvesting and shifts in excitation energy between photosystem I and II. In contrast, UWO 241 exhibited high rates of PSI-driven cyclic electron flow (CEF) and a larger capacity for nonphotochemical quenching (NPQ). Furthermore, UWO 241 exhibited constitutively high activity of two key ascorbate cycle enzymes, ascorbate peroxidase and glutathione reductase and maintained a large ascorbate pool. These results matched the ability of the psychrophile to maintain low ROS under short-term photoinhibition conditions. We conclude that tight control over photostasis and ROS levels are essential for photosynthetic life to flourish in a native habitat of permanent photooxidative stress. We propose to rename this organism Chlamydomonas priscuii.
... There may be cooperation or complementarity between photosynthetic respiratory metabolism and NPQ in order to maintain oxidative balance within cells. Some other photoprotective mechanisms closely related to NPQ also have been identified, such as the water-water cycle and periodic electron transport and in regulating NPQ induction [56][57][58]. The value of NPQ increased continuously with increasing time or light intensity under both dark and light acclimation conditions. ...
In recent years, much effort has been devoted to understanding the response of plants to various light sources, largely due to advances in industry light-emitting diodes (LEDs). In this study, the effect of different light modes on rocket (Eruca sativa. Mill.) photosynthetic performance and other physiological traits was evaluated using an orthogonal design based on a combination between light intensity, quality, and photoperiod factors. Some morphological and biochemical parameters and photosynthetic efficiency of the plants were analyzed. Plants grew in a closed chamber where three light intensities (160, 190, and 220 μmol m-2 s-1) provided by LEDs with a combination of different ratios of red, green, and blue (R:G:B- 7:0:3, 3:0:7, and 5:2:3) and three different photoperiods (light/dark -10/14 h, 12/12 h, and 14/10 h) were used and compared with white fluorescent light (control). This experimental setup allowed us to study the effect of 9 light modes (LM) compared to white light. The analyzes performed showed that the highest levels of chlorophyll a, chlorophyll b, and carotenoids occurred under LM4, LM3, and LM1, respectively. Chlorophyll a fluorescence measurement showed that the best effective quantum yield of PSII photochemistry Y(II), non-photochemical quenching (NPQ), photochemical quenching coefficient (qP), and electron transport ratio (ETR) were obtained under LM2. The data showed that the application of R7:G0:B3 light mode with a shorter photoperiod than 14/10 h (light/dark), regardless of the light intensity used, resulted in a significant increase in growth as well as higher photosynthetic capacity of rocket plants. Since, a clear correlation between the studied traits under the applied light modes was not found, more features should be studied in future experiments.
... The absolute pH dependency of quenching in P. tricornutum (apparent pKa of 4.7, Figure 1C) was shifted towards more acidic values than in green algae (apparent pKa of 6.2 in Chlamydomonas reinhardtii, (Tian et al., 2019)). Thus, quenching could reflect photosystem II (PSII) photodamage, which is expected at such low lumen pH values (Krieger and Weis, 1993;Spetea et al., 1997;Kramer et al., 2003). However, we found a very similar Stern-Volmer (S-V) relationship between NPQ and PSII photochemical quantum yield (Fv'/Fm') during a HL to LL relaxation and the acid to neutral pH transition (Supplementary Figure S1). ...
Diatoms are amongst the most successful clades of oceanic phytoplankton, significantly contributing to photosynthesis on Earth. Their ecological success likely stems from their ability 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 of via Non Photochemical Quenching (NPQ). While the molecular effectors of this process, as well as the role of the Proton Motive Force (PMF) in its regulation are known, the putative regulators of the PET/PMF relationship in diatoms remain unidentified. Here, we demonstrate that the H ⁺ /K ⁺ antiporter KEA3 is the main regulator of the coupling between PMF and PET in the model diatom Phaeodactylum tricornutum . By controlling the PMF, it modulates NPQ responses at the onset of illumination, during transients and in steady state conditions. Under intermittent light KEA3 absence results in reduced fitness. Using a parsimonious model including only two components, KEA3 and the diadinoxanthin de-epoxidase, we can describe most of the feedback loops observed between PET and NPQ. This two-components 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 Ca ²⁺ -binding domain in KEA3 that controls its activity. This circuit is likely finely tuned by the NPQ effector proteins LHCX, providing diatoms with the required flexibility to thrive in different ocean provinces.
One sentence summary
The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( https://academic.oup.com/plcell/pages/General-Instructions ) is Giovanni Finazzi.
... First, the suppression of electron transport at the cytochrome (Cyt) b 6 f complex contributes to P700 oxidation. The mechanism for this is either through a difference in the proton concentration across thylakoid membrane (ΔpH)dependent mechanism, by the so-called photosynthetic control (Foyer et al., 1990;Kramer et al., 2003), or through the proposed mechanism dependent on reduced plastoquinone (PQ) pool, termed as RISE . Second, an electron sink is required as a prerequisite for P700 oxidation. ...
Against the potential risk in oxygenic photosynthesis, that is, the generation of reactive oxygen species, photosynthetic electron transport needs to be regulated in response to environmental fluctuations. One of the most important regulations is keeping the reaction center chlorophyll (P700) of photosystem I in its oxidized form in excess light conditions. The oxidation of P700 is supported by dissipating excess electrons safely to O2, and we previously found that the molecular mechanism of the alternative electron sink is changed from flavodiiron proteins (FLV) to photorespiration in the evolutionary history from cyanobacteria to plants. However, the overall picture of the regulation of photosynthetic electron transport is still not clear in bryophytes, the evolutionary intermediates. Here, we investigated the physiological roles of FLV and photorespiration for P700 oxidation in the liverwort Marchantia polymorpha by using the mutants deficient in FLV (flv1) at different O2 partial pressures. The effective quantum yield of photosystem II significantly decreased at 2kPa O2 in flv1, indicating that photorespiration functions as the electron sink. Nevertheless, it was clear from the phenotype of flv1 that FLV was dominant for P700 oxidation in M. polymorpha. These data suggested that photorespiration has yet not replaced FLV in functioning for P700 oxidation in the basal land plant probably because of the lower contribution to lumen acidification, compared with FLV, as reflected in the results of electrochromic shift analysis.
... Photosynthetic organisms utilize FoF1 ATP synthase (FoF1) for a solar-to-chemical energy conversion system to produce ATP, the universal energy currency for cells. Under illumination, photosynthetic electron transport is activated, generating a proton electrochemical gradient (proton motive force, pmf) across the thylakoid membrane, which drives ATP synthesis (1)(2)(3)(4). In the dark, or when the pmf is insufficient, FoF1 hydrolyzes ATP as the reverse reaction and transports H + to the lumen. ...
The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP-hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.
... Therefore, the size of ΔpH may be partly compensated by increasing the partitioning of ΔpH in pmf in pgr5 (Yamamoto et al., 2016). To move from Δψ to ΔpH, some cations (mainly Mg 2+ and K + ) have to be transported to the stroma via the thylakoid membrane (Kramer et al., 2003). Hind et al. (1974) indicated that the outflow of these cations could facilitate pmf adjusting into the ΔpH form. ...
The cyclic electron transport (CET), after the linear electron transport (LET), is another important electron transport pathway during the light reactions of photosynthesis. The proton gradient regulation 5 (PGR5)/PRG5-like photosynthetic phenotype 1 (PGRL1) and the NADH dehydrogenase-like complex pathways are linked to the CET. Recently, the regulation of CET around photosystem I (PSI) has been recognized as crucial for photosynthesis and plant growth. Here, we summarized the main biochemical processes of the PGR5/PGRL1-dependent CET pathway and its physiological significance in protecting the photosystem II and PSI, ATP/NADPH ratio maintenance, and regulating the transitions between LET and CET in order to optimize photosynthesis when encountering unfavorable conditions. A better understanding of the PGR5/PGRL1-mediated CET during photosynthesis might provide novel strategies for improving crop yield in a world facing more extreme weather events with multiple stresses affecting the plants.
... Whereas in respiratory membranes the pmf is mainly stored as Δψ, for a long time it was assumed that the Δψ under steady-state illumination is small in photosynthetic thylakoid membranes and the pmf consists mainly of ΔpH 5 . This view of thylakoid pmf storage has been challenged by improved spectroscopic analyses [6][7][8][9] that disentangle the contributions of ΔpH and Δψ to total pmf storage, combined with the identification and characterization of thylakoid ion channel/transporters mutants, implicating storage of a fraction of pmf as Δψ. An explanation of the controversy about pmf partitioning in thylakoid membranes is that the ΔpH/Δψ ratio is not static but dynamic and could depend on environmental/metabolic conditions in vivo (for example ref. 10 ), as well as the experimental conditions under which it is measured 8 . ...
In photosynthetic thylakoid membranes the proton motive force (pmf) not only drives ATP synthesis, in addition it is central to controlling and regulating energy conversion. As a consequence, dynamic fine-tuning of the two pmf components, electrical (Δψ) and chemical (ΔpH), is an essential element for adjusting photosynthetic light reactions to changing environmental conditions. Good evidence exists that the Δψ/ΔpH partitioning is controlled by thylakoid potassium and chloride ion transporters and channels. However, a detailed mechanistic understanding of how these thylakoid ion transporter/channels control pmf partitioning is lacking. Here, we combined functional measurements on potassium and chloride ion transporter and channel loss-of-function mutants with extended mathematical simulations of photosynthetic light reactions in thylakoid membranes to obtain detailed kinetic insights into the complex interrelationship between membrane energization and ion fluxes across thylakoid membranes. The data reveal that potassium and chloride fluxes in the thylakoid lumen determined by the K+/H+ antiporter KEA3 and the voltage-gated Cl− channel VCCN1/Best1 have distinct kinetic responses that lead to characteristic and light-intensity-dependent Δψ/ΔpH oscillations. These oscillations fine-tune photoprotective mechanisms and electron transport which are particularly important during the first minutes of illumination and under fluctuating light conditions. By employing the predictive power of the model, we unravelled the functional consequences of changes in KEA3 and VCCN1 abundance and regulatory/enzymatic parameters on membrane energization and photoprotection. Thylakoid membranes must maintain a balance of their electrical and pH potentials, created by light-driven photosynthesis. Photoprotective mechanisms and electron transport are fine-tuned through pH oscillations mediated by K+ and Cl– channels.
... Photosynthetic electron transport is coupled with proton translocation across the thylakoid membrane, resulting in the formation of transmembrane H + concentration (ΔpH) and electrical potential (ΔΨ) gradients. Although both ΔpH and Δψ contribute to ATP synthesis as a proton motive force (pmf), only the ΔpH component can activate the PsbS-and xanthophyll-cycle-dependent NPQ while down-regulating electron transport during the plastoquinol oxidation step at the cytochrome b 6 /f complex (photosynthetic control, Kramer et al. 2003;Yamori and Shikanai 2016). Recent evidence suggests that several ion channels, such as the thylakoid K + channel TPK3, K + efflux antiporter KEA3, and Cl − channel Best1/VCCN1, adjust electron transport and functions in photoprotective mechanisms (Figs. 5 and 7;Carraretto et al. 2013;Kunz et al. 2014;Duan et al. 2016;Herdean et al. 2016). ...
Crop productivity would have to increase by 60–110% compared with the 2005 level by 2050 to meet both the food and energy demands of the growing population. Although more than 90% of crop biomass is derived from photosynthetic products, photosynthetic improvements have not yet been addressed by breeding. Thus, it has been considered that enhancing photosynthetic capacity is considered a promising approach for increasing crop yield. Now, we need to identify the specific targets that would improve leaf photosynthesis to realize a new Green Revolution. This chapter summarizes the various genetic engineering approaches that can be used to enhance photosynthetic capacity and crop productivity. The targets considered for the possible candidates include Rubisco, Rubisco activase, enzymes of the Calvin–Benson cycle, and CO2 transport, as well as photosynthetic electron transport. Finally, it describes the importance of considering ways to improve photosynthesis not under the stable environmental conditions already examined in many studies with the aim of improving photosynthetic capacity, but under natural conditions in which various environmental factors, and especially irradiation, continually fluctuate.
... pH is the trigger for qE activation, a direct measure of lumenal pH is not straightforward and there is still debate on the actual lower limit range [31][32][33]. Several authors claim that the pH of the lumen cannot go below pH 5.5 or even 6.0 [34,35]. However, most of in vitro quenching experiments are commonly performed between pH 5.0 and 3.5 (see, for instance, [25,36]), and little is known on the organization and size of the particles obtained at these pH conditions. ...
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.
... In particular, copper, which is present in higher amounts in synthetic wastewater than in half-strength Hutner's (5 and 0.12 μM, respectively), can inhibit phosphorylation by ATP synthase (Uribe and Stark 1982;Maksymiec 1998). A consequence of impeding ATP generation is the build-up of a proton gradient across the thylakoid lumen (Kramer et al. 2003). In turn, this gradient causes non-photochemical quenching, Y(NPQ), to be increased through the xanthophyll cycle (Horton et al. 1996;Li et al. 2002), as was observed in this study. ...
Lemnaceae, i.e. duckweed species, are attractive for phytoremediation of wastewaters, primarily due to their rapid growth, high nutrient uptake rates, tolerance to a broad range of growing conditions and ability to expeditiously assimilate a variety of pollutants. Light is essential for plant growth, and therefore, phytoremediation. Nevertheless, the effect of light intensity remains poorly understood in relation to phytoremediation, a knowledge gap that impedes the development of indoor, fully controlled, stacked remediation systems. In the present study, the effect of light intensity (10–850 μmol m⁻² s⁻¹) on the phytoremediation potential of Lemna minor was assessed. Plants were grown on either an optimal growth medium (half-strength Hutner’s) or synthetic dairy processing wastewater, using stationary axenic (100 mL) or re-circulating non-sterile (11.7 L) systems. The relative growth rate (RGR) of L. minor grown on half-strength Hutner’s increased proportionally with increasing light intensity. In contrast, the RGR of L. minor grown on synthetic dairy wastewater did not increase with light over an intensity range from 50 to 850 μmol m⁻² s⁻¹. On synthetic dairy wastewater, total nitrogen and total phosphorous removal also remained unchanged between 50 and 850 μmol m⁻² s⁻¹, although L. minor protein content (% fresh weight) increased from 1.5 to 2% at higher light intensities. Similar results were obtained with the larger re-circulating system. The results demonstrate interactive effects of light intensity and wastewater composition on growth and phytoremediation potential of L. minor. The data imply that light intensities above 50 μmol m⁻² s⁻¹ may not necessarily confer benefits in duckweed wastewater remediation, and this informs engineering of stacked, indoor remediation systems.
... Interestingly, this mutation affected the phenotype both qualitatively and quantitatively. Generally, a decrease in ATP synthase leads to the accumulation of H + in the lumenal space of thylakoid membranes, that is, increases pmf (Kanazawa et al., 2017;Kramer, Cruz, & Kanazawa, 2003;Takizawa, Cruz, Kanazawa, & Kramer, 2007;Takizawa, Kanazawa, & Kramer, 2008). Enhancing gH + decreased pmf, as revealed by ΔpH formation across thylakoid membranes under high light intensity (Takagi et al., 2017), as well as suppressed P700 oxidation in PSI. ...
The main production site of reactive oxygen species (ROS) in photosynthetic organisms is photosystem (PS) I of thylakoid membranes. Unless the suppression mechanism of ROS production functions, PSI easily suffers from oxidative damages by ROS attack. Miyake group has elucidated the production and suppression mechanisms of ROS in PSI. The reaction center chlorophyll, P700, in PSI functions in P700 photo-oxidation reduction cycle. The photoexcited P700, P700*, can donate electron to O2 producing superoxide radical, ROS, with oxidized to P700⁺. The accumulation of the P700⁺ decreases the probability of the presence of P700* not to produce ROS. The present review describes the molecular mechanism to oxidize P700 and to accumulate P700⁺ in PSI. Tight coupling between the light and the dark reactions in photosynthesis accumulates H⁺ in the luminal side and electron in plastoquinone pool of thylakoid membranes on exposure to the environmental stress, which lowers the electron transport activity of Cyt b6/f-complex and suppresses the electron flux to PSI with P700⁺ accumulated. We discuss the molecular mechanisms to accumulate e⁻ and H⁺ and its relationship with ATP synthase activity from the aspect of P700 oxidation in PSI.
Photosystem II (PSII) of the photosynthetic apparatus in oxygenic organisms contains a catalytic center that performs one of the most important reactions in bioenergetics: light-dependent water oxidation to molecular oxygen. The catalytic center is a Mn4CaO5 cluster consisting of four cations of manganese and one calcium cation linked by oxygen bridges. The authors reported earlier that a structural transition occurs at pH 5.7 in the cluster resulting in changes in manganese cation(s) redox potential and elevation of the Mn‑clus-ter resistance to reducing agents. The discovered effect was examined in a series of investigations that are reviewed in this work. It was found that, at pH 5.7, Fe(II) cations replace not two manganese cations as it happens at pH 6.5 but only one cation; as a result, a chimeric Mn3Fe1 cluster is produced. In the presence of exogenous calcium ions, membrane preparations of PSII with such a chimeric cluster are capable of evolving oxygen in the light (at a rate of approximately 25% of the rate in native PSII). It was found that photoinhibition that greatly depends on the processes of oxidation or reduction at pH 5.7 slows down as compared with pH 6.5. PSII preparations were also more resistant to thermal inactivation at pH 5.7 than at pH 6.5. However, in PSII preparations lacking manganese cations in the oxygen-evolving complex, the rates of photoinhibition at pH 6.5 and 5.7 did not differ. In thylakoid membranes, protonophores that abolish the proton gradient and increase pH in the lumen (where the manganese cluster is located) from 5.7 to 7.0 considerably elevated the rate of PSII photoinhibition. It is assumed that the structural transition in the Mn-cluster at pH 5.7 is involved in the mechanisms of PSII defense against photoinhibition.
Plant yields heavily depend on proper macro‐ and micronutrient supply from the soil. In the leaf cells, nutrient ions fulfill specific roles in biochemical reactions, especially photosynthesis housed in the chloroplast. Here, a well‐balanced ion homeostasis is maintained by a number of ion transport proteins embedded in the envelope and thylakoid membranes. Ten years ago, the first alkali metal transporters from the K⁺ EFFLUX ANTIPORTER family were discovered in the model plant Arabidopsis. Since then, our knowledge about the physiological importance of these carriers and their substrates has greatly expanded. New insights into the role of alkali ions in plastid gene expression and photoprotective mechanisms, both prerequisites for plant productivity in natural environments, were gained. The discovery of a Cl⁻ channel in the thylakoid and several additional plastid alkali and alkali metal transport proteins have advanced the field further. Nevertheless, scientists still have long ways to go before a complete systemic understanding of the chloroplast's ion transportome will emerge. In this Tansley review, we highlight and discuss the achievements of the last decade. More importantly, we make recommendations on what areas to prioritize, so the field can reach the next milestones. One area, laid bare by our similarity‐based comparisons among phototrophs is our lack of knowledge what ion transporters are used by cyanobacteria to buffer photosynthesis fluctuations.
The proton motive force (pmf) generated across the thylakoid membrane rotates the Fo-ring of ATP synthase in chloroplasts. The pmf comprises two components: membrane potential (∆Ψ) and proton concentration gradient (∆pH). Acidification of the thylakoid lumen resulting from ∆pH downregulates electron transport in the cytochrome b6f complex. This process, known as photosynthetic control, is crucial for protecting photosystem I (PSI) from photodamage in response to fluctuating light. To optimize the balance between efficient photosynthesis and photoprotection, it is necessary to regulate pmf. Cyclic electron transport around PSI and pseudo-cyclic electron transport involving flavodiiron proteins contribute to the modulation of pmf magnitude. By manipulating the ratio between the two components of pmf, it is possible to modify the extent of photosynthetic control without affecting the pmf size. This adjustment can be achieved by regulating the movement of ions (such as K+ and Cl−) across the thylakoid membrane. Since ATP synthase is the primary consumer of pmf in chloroplasts, its activity must be precisely regulated to accommodate other mechanisms involved in pmf optimization. Although fragments of information about each regulatory process have been accumulated, a comprehensive understanding of their interactions is lacking. Here, I summarize current knowledge of the network for pmf regulation, mainly based on genetic studies.
Like all organisms performing oxygenic photosynthesis, Chlamydomonas captures light energy in two photochemical steps to drive linear electron flow from water to NADPH and to produce ATP. However, this process alone is not sufficient to drive CO2 fixation in the Calvin–Benson cycle and to respond to environmental and metabolic constraints, for example, light availability or metabolic needs in term of ATP and NADPH. A complex network of alternative electron flows, comprises cyclic electron flow around photosystem I and various water-to-water cycles that utilize O2 as an alternative electron acceptor, provide an additional degree of freedom to face this challenge. The present chapter describes the various alternative routes of photosynthetic electron transfer in Chlamydomonas, describes how they are coordinated for optimization of photosynthesis and gives a retrospective on the early physiology work on those alternative electron flows among model photosynthetic organisms.
Chapter 6 of Marschner's Mineral Nutrition of Plants, Fourth Edition. This book presents sections on the uptake and transport of nutrients in plants, root-shoot interactions, the role of mineral nutrition in yield formation, stress physiology, water relations, functions of mineral nutrients and contribution of plant nutrition to nutritional quality and global nutrition security of human populations.
Other sections focus on the effects of external and internal factors on root growth, rhizosphere chemistry and biology, and nutrient cycling. In addition, this updated edition includes color figures and a new chapter on the impacts of climate change on soil fertility and crop nutrition. An understanding of the mineral nutrition of plants is of fundamental importance in both basic and applied plant sciences. The fourth edition of this book retains the aim of the first in presenting the principles of mineral nutrition in the light of current advances.
Adenosine triphosphate (ATP) is a central metabolite that functions as the energy currency in a living cell. Therefore, visualizing cellular ATP dynamics provides the fundamental information necessary to understand the molecular events involving life phenomena. Live cell imaging technologies using fluorescence (FL)-based indicators have been developed to analyze the dynamics of various biological processes, such as intracellular ATP synthesis and consumption. However, the application of FL-based indicators to plant cells is limited due to the presence of strong chlorophyll autofluorescence, which drastically worsen the signal-to-noise ratio. The bioluminescent (BL) indicators that do not require excitation light could overcome this problem. In this chapter, we introduce a methodology to analyze ATP dynamics in plant cells using BL ATP indicators.
Inappropriate agricultural practices and environmental impacts are worsening soil salinity. This affects crop yield and, consequently, the dynamics of the international market and food security. According to the stage of development of the plant, the duration of exposure, and the intensity of stress, different responses are triggered to maintain vital metabolic reactions and the integrity of cellular components. The most consumed crops in the world, in general, are glycophytes, and the efforts to find salt-tolerant cultivars have not yet resulted in wide practical application in the field. Since halophytic plants can complete their life cycle under highly saline conditions, they can provide clues about pathways to be explored to improve glycophytes’ response to salinity. In this context, the search for differences between glycophytes and halophytes has contributed to the identification of promising traits of the latter that can enable the achievement of the mentioned aim. Among them, the existence of transcripts unique to halophytes and unannotated, therefore, with unknown functions. Furthermore, although responses to salt are generally common between these two groups of plants, halophytes succeed, for example, regarding the balancing of the Na+/K+ ratio. It can occur through the ability to compartmentalize higher levels of Na+ in vacuoles and to maintain or distribute K+ more efficiently. Moreover, other highlights that can be explored include the ions usage for osmotic adjustment as a metabolically cheaper alternative and more powerful antioxidant system and stress signaling pathways.
During the light-dependent reaction of photosynthesis green plants couple photoinduced cascades of redox reactions with transmembrane proton translocations to generate reducing equivalents and chemical energy in the form of NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate), respectively. We mimic these basic processes by combining molecular ruthenium polypyridine-based photocatalysts and inverted vesicles derived from Escherichia coli . Upon irradiation with visible light, the interplay of photocatalytic nicotinamide reduction and enzymatic membrane-located respiration leads to the simultaneous formation of two biologically active cofactors, NADH (nicotinamide adenine dinucleotide) and ATP, respectively. This inorganic-biologic hybrid system thus emulates the cofactor delivering function of an active chloroplast
Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150–170 mV; to achieve in vivo rates the imposed pmf must reach 200 mV. The key question then is ‘does the pmf generated by electron transport exceed 200 mV, or even 170 mV?’ The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.
Astaxanthin is a high-valued ketocarotenoid that is rarely synthesized in higher plants due to their lack of a carotenoid ketolase. Overexpression of a heterologous β-carotene ketolase gene coupled with other pathway genes in higher plants has triggered the production of various ketocarotenoids in the transformants. Here we reported that constitutive and simultaneous overexpression of four genes involved in astaxanthin biosynthesis in Brassica napus resulted in the accumulation of the nonnative ketocarotenoids such as astaxanthin and ketolutein, and the decreased production of chlorophylls, lutein and violaxanthin in leaves. As a result, transgenic plants showed lower non-photochemical quenching (NPQ) under high light, leading to moderate photoinhibition of photosystem II (PSII). Such PSII photoinhibition depressed linear electron transport and thus restricted the rate of CO2 assimilation. Consequently, transgenic plants grew slower and showed less biomass than wild type. These results indicated that endogenous synthesis of ketocarotenoids negatively affected photosynthesis and impaired plant growth in B. napus. Therefore, to prevent the side effect on normal growth, seed specific expression of the transgenes may be necessary for enhanced production of astaxanthin in seeds.
Thylakoids are flattened sacs isolated from other membranes; cristae are attached to the rest of the inner mitochondrial membrane by the crista junction, but the crista lumen is separated from the intermembrane space. The shape of thylakoids and cristae involves membranes with small (5 - 30 nm) radii of curvature. While the mechanism of curvature is not entirely clear, it seems to be largely a function of Curt proteins in thylakoids and Mitochondrial Organising Site and Crista Organising Centre proteins and oligomeric FOF1 ATP synthase in cristae. A subordinate, or minimal, role is attributable to lipids with areas of their head group area greater (convex leaflet) or smaller (concave leaflet) than the area of the lipid tail; examples of the latter group are monogalactosyldiglyceride in thylakoids and cardiolipin in cristae. The volume per unit area on the lumen side of the membrane is less than that of the chloroplast stroma or cyanobacterial cytosol for thylakoids, and mitochondrial matrix for cristae. A low volume per unit area of thylakoids and cristae means a small lumen width that is the average of wider spaces between lipid parts of the membranes and the narrower gaps dominated by extra-membrane components of transmembrane proteins. These structural constraints have important implications for the movement of the electron carriers plastocyanin and cytochrome c6 (thylakoids) and cytochrome c (cristae) and hence the separation of the membrane-associated electron donors to, and electron acceptors from, these water-soluble electron carriers. The donor/acceptor pairs, are the cytochrome fb6Fenh complex and P700⁺ in thylakoids, and Complex III and Complex IV of cristae. The other energy flux parallel to the membranes is that of the proton motive force generated by redox-powered H⁺ pumps into the lumen to the proton motive force use in ATP synthesis by H⁺ flux from the lumen through the ATP synthase. For both the electron transport and proton motive force movement, concentration differences of reduced and oxidised electron carriers and protonated and deprotonated pH buffers are involved. The need for diffusion along a congested route of these energy transfer agents may limit the separation of sources and sinks parallel to the membranes of thylakoids and cristae.
The rate of ATP synthesis catalyzed by normal and by dithiothreitol-modified ATPases is investigated as a function of ΔpH in spinach chloroplasts at constant pHout. The transmembrane ΔpH was generated by an acid-base transition and the reaction time was limited to 150 ms by using a rapidly mixing quenched-flow apparatus. The result was that the functional dependence of the rate on ΔpH is shifted to lower ΔpH values and that the shape of this curve is altered after dithiothreitol modification. The maximal rate (400 ATP / CF1 per s) is the same under both conditions.
Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.
This review describes and assesses pathways likely to influence and stabilize the ATP/reductant balance during whole cell
photosynthesis. The sole reductive step of the Calvin cycle occurs during the conversion of 3‐phosphoglycerate to triose phosphate.
Photophosphorylation linked to this reaction can undoubtedly supply most of the ATP required by the Calvin cycle and other
chloroplastic reactions. Small but crucial contributions must come from several other pathways, some of which involve co‐operation
between the chloroplast and the rest of the cell. Extrachloroplastic compartments can contribute to chloroplastic ATP requirements
by supplying ATP directly or, probably more significantly, by accepting reducing equivalents and so supporting ATP synthesis
within the chloroplast.
A noninvasive technique is introduced with which relative proton to electron stoichiometries (H(+)/e(-) ratios) for photosynthetic electron transfer can be obtained from leaves of living plants under steady-state illumination. Both electron and proton transfer fluxes were estimated by a modification of our previously reported dark-interval relaxation kinetics (DIRK) analysis, in which processes that occur upon rapid shuttering of the actinic light are analyzed. Rates of turnover of linear electron transfer through the cytochrome (cyt) b(6)f complex were estimated by measuring the DIRK signals associated with reduction of cyt f and P(700). The rates of proton pumping through the electron transfer chain and the CF(O)-CF(1) ATP synthase (ATPase) were estimated by measuring the DIRK signals associated with the electrochromic shifting of pigments in the light-harvesting complexes. Electron transfer fluxes were also estimated by analysis of saturation pulse-induced changes in chlorophyll a fluorescence yield. It was shown that the H(+)/e(-) ratio, with respect to both cyt b(6)f complex and photosystem (PS) II turnover, was constant under low to saturating illumination in intact tobacco leaves. Because a H(+)/e(-) ratio of 3 at a low light is generally accepted, we infer that this ratio is maintained under conditions of normal (unstressed) photosynthesis, implying a continuously engaged, proton-pumping Q cycle at the cyt b(6)f complex.
Nonphotochemical quenching (NPQ) of excitation energy, which protects higher plant photosynthetic machinery from photodamage, is triggered by acidification of the thylakoid lumen as a result of light-induced proton pumping, which also drives the synthesis of ATP. It is clear that the sensitivity of NPQ is modulated in response to changing physiological conditions, but the mechanism for this modulation has remained unclear. Evidence is presented that, in intact tobacco or Arabidopsis leaves, NPQ modulation in response to changing CO(2) levels occurs predominantly by alterations in the conductivity of the CF(O)-CF(1) ATP synthase to protons (g(H)(+)). At a given proton flux, decreasing g(H)(+) will increase transthylakoid proton motive force (pmf), thus lowering lumen pH and contributing to the activation of NPQ. It was found that an approximately 5-fold decrease in g(H)(+) could account for the majority of NPQ modulation as atmospheric CO(2) was decreased from 2,000 ppm to 0 ppm. Data are presented that g(H)(+) is kinetically controlled, rather than imposed thermodynamically by buildup of DeltaG(ATP). Further results suggest that the redox state of the ATP synthase gamma-subunit thiols is not responsible for altering g(H)(+). A working model is proposed wherein g(H)(+) is modulated by stromal metabolite levels, possibly by inorganic phosphate.
High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly 'photoinhibited', leaves. Functional implications of these observations are discussed.
Spinach (Spinacia oleracea var "Yates") plants in hydroponic culture were exposed to stepwise increased concentrations of NaCl or NaNO(3) up to a final concentration of 300 millimoles per liter, at constant Ca(2+)-concentration. Leaf cell sap and extracts from aqueously isolated spinach chloroplasts were analyzed for mineral cations, anions, amino acids, sugars, and quarternary ammonium compounds. Total osmolality of leaf sap and photosynthetic capacity of leaves were also measured. For comparison, leaf sap from salt-treated pea plants was also analyzed. Spinach plants under NaCl or NaNO(3) salinity took up large amounts of sodium (up to 400 millimoles per liter); nitrate as the accompanying anion was taken up less (up to 90 millimoles per liter) than chloride (up to 450 millimoles per liter). Under chloride salinity, nitrate content in leaves decreased drastically, but total amino acid concentrations remained constant. This response was much more pronounced (and occurred at lower salt concentrations) in leaves from the glycophyte (pea, Pisum sativum var "Kleine Rheinländerin") than from moderately salt-tolerant spinach. In spinach, sodium chloride or nitrate taken up into leaves was largely sequestered in the vacuoles; both salts induced synthesis of quarternary ammonium compounds, which were accumulated mainly in chloroplasts (and cytosol). This prevented impairment of metabolism, as indicated by an unchanged photosynthetic capacity of leaves.
Ion-sensitive microelectrodes were used to measure Cl(-) and H(+) activities in the cytoplasm of the unicellular green alga Eremosphaera viridis de Bary. In the light, cytoplasmic Cl(-) activity was 2.2 millimolar at most and cytoplasmic H(+) activity was about 5.4.10(-8) molar (pH 7.3). Darkening resulted in a permanent increase of the Cl(-) activity to 3.2 millimolar and in a transient acidification, which was compensated within 3 to 5 minutes. Switching light on again decreased the Cl(-) activity to the light level (2.2 millimolar). Simultaneously, a transient alkalization of the cytoplasm was observed. The transient character of the light-dependent pH changes was probably caused by pH-stat mechanisms, whereas the light-dependent Cl(-) activity changes were compensated to a much smaller degree. Studies with different inhibitors (3-(3,4-dichlorophenyl)-1, 1-dimethylurea, piretanide, venturicidin) indicated a direct relation between the light-driven H(+) flow across the thylakoid membrane and the observed light-dependent Cl(-) and H(+) activity changes in the cytoplasm. It is suggested that light-driven H(+) flux across the thylakoid membrane was in part electrically compensated by a parallel Cl(-) flux. The resulting Cl(-) and H(+) activity changes in the stroma were compensated by Cl(-) and H(+) fluxes across the chloroplast envelope giving rise to the observed Cl(-) and H(+) activity changes in the cytoplasm.
A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.
Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially `pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.
Proton motive force (pmf), established across the thylakoid membrane by photosynthetic electron transfer, functions both to drive the synthesis of ATP and initiate processes that down-regulate photosynthesis. At the same time, excessively low lumen pH can lead to the destruction of some lumenal components and sensitization of the photosynthetic apparatus to photoinhibition. Therefore, in order to understand the energy budget of photosynthesis, its regulation and responses to environmental stresses, it is essential to know the magnitude of pmf, its distribution between ΔpH and the electric field (Δ) as well as the relationships between these parameters and ΔG_ATP, and down-regulatory and inhibitory processes. We review past estimates of lumen pH and propose a model that can explain much of the divergent data in the literature. In this model, in intact plants under permissive conditions, photosynthesis is regulated so that lumen pH remains moderate (between 5.8 and 6.5), where it modulates the activity of the violaxanthin deepoxidase, does not significantly restrict the turnover of the cytochrome b_6f complex, and does not destabilize the oxygen evolving complex. Only under stressed conditions, where light input exceeds the capacity of both photosynthesis and down-regulatory processes, does lumen pH decrease below 5, possibly contributing to photoinhibition. A value of n = 4 for the stoichiometry of protons pumped through the ATP synthase per ATP synthesized, and a minor contribution of Δ to pmf, will allow moderate lumen pH to sustain the observed levels of ΔG_ATP.
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 responsive 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 (ApH) 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 cone 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 Chl 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.
The kinetics of the light-induced changes in the electrical potential across the thylakoid membranes of intact chloroplasts of Peperomia metallica were measured in the presence of ionophores A23187, volinomycin and nigericin, by means of micro-cappillary glass electrodes. A slow increase in potential occurs in the light in the presence of A23187 and of volinomycin after the completion of the well-known phase 1 and phase 2 potential response. This increase is inhibited by nigericin. The results are discussed to be due to a membrane diffusion potential associated with a K+-concentration gradient across the thylakoid membrane. This gradient is likely to be formed due to the passive efflux of K+-ions occurring across the thylakoid membranes in conjunction with the electrogenic proton influx.
The observed levels of DeltaG(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid proton motive force (pmf) equivalent to a Delta pH of similar to2.5 units. If as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than similar to5. In contrast, we have presented evidence that the pH of the thylakoid lumen does not drop below pH similar to5.8 [Kramer, D. M., Sacksteder, C. A., and Cruz, J. A. (1999) Photosynth. Res. 60, 151-163], leading us to propose that Delta psi can contribute to steady-state pmf: In this work, it is demonstrated, through assays on isolated thylakoids and computer simulations, that thylakoids can store a substantial fraction of pmf as Delta psi, provided that the activities of ions permeable to the thylakoid membrane in the chloroplast stromal compartment are relatively low and the buffering capacity (beta) for protons of the lumen is relatively high. Measurements of the right-induced electrochromic shift (ECS) confirm the ionic strength behavior of steady-state Delta psi in isolated, partially uncoupled thylakoids. Measurements of the ECS in intact plants illuminated for 65 s were consistent with low concentrations of permeable ions and similar to 50% storage of pmf as Delta psi. We propose that the plant cell, possibly at the level of the inner chloroplast envelope, can control the parsing of pmf into Delta psi and Delta pH by regulating the ionic strength and balance of the chloroplast. In addition, this work demonstrates that, under certain conditions, the kinetics of the light-induced ECS can be used to estimate the fractions of pmf stored as Delta psi and Delta pH both in vitro and in vivo.
We have investigated the ATP synthesis associated with acid-base transitions in chloroplast lamellae under conditions which allow simultaneous control of the thermodynamic variables, ΔpH, membrane potential and ΔGATP. These variables have been directly imposed rather than simply inferred. Since the initiation of labeled Pi incorporation seems to measure accurately the initiation of net ATP synthesis, the following conclusions can be drawn: (1) The proton-motive force which is just sufficient for ATP synthesis provides almost exactly the required energy for ΔGATP if the efflux of three H+ is required for each ATP molecule formed. (2) The membrane potential and the ΔpH contribute to the proton-motive force in a precisely additive way. Thus, the threshold can be reached or exceeded by a ΔpH in the absence of a membrane potential, by a membrane potential in the absence of a ΔpH, or by any combination of membrane potential and ΔpH. With a large enough membrane potential, ATP synthesis occurs even against a small inverse ΔpH. In each instance the combined ΔpH and membrane potential necessary for initiation of ATP synthesis represent the same threshold proton-motive force.
The use of solar energy in photosynthesis depends on the ability to safely dissipate excess energy. The key dissipation process employed by plants in their natural environment is mediated by a particular group of carotenoids. Multiple levels of control allow adjustments in energy dissipation activity in response to changing levels of light stress in the natural environment. Recent advances in the understanding of the photophysics, biochemical regulation and ecophysiology of this essential photoprotective process are reviewed.
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.
Field-indicating absorption changes have been measured in mutant strains of Chlorella sorokiniana and Chlamydomonas reinhardtii lacking one or several chlorophyll-protein complexes. Using mutants which lack PS II centers and most of the chlorophyll antenna, we could characterize two types of probe, with linear or quadratic response to the membrane potential. The probes with linear response present an electrochromic spectrum with maxima at 514 and 486 nm and a minimum at 472 nm; those which respond quadratically present a spectrum with maxima at 464 and 504 nm and a minimum at 479 nm. By measuring the relative contribution of these probes upon a weak actinic flash, the offset of the membrane potential may be estimated under various experimental conditions. In anaerobiosis in the dark, a large permanent membrane potential arises from the hydrolysis of ATP, mainly of mitochondria! origin. We have also analyzed the electrochromic absorption changes in other mutant strains lacking either PS II only,or PS I and the major fraction of light-harvesting complexes. The quadratic probes are present to a similar extent in every strain investigated, which suggests that they are not associated with any of the major chlorophyll-protein complexes. These probes are also conserved in higher plants. In contrast, the linear electrochromic changes are roughly proportional to the overall amount of chlorophyll, either associated with the photocenter or with the antenna.
Carotenoids are components of every pigment-protein complex in the photosynthetic apparatus of higher plants. These pigments,
previously referred to as ‘accessory,’ are now recognized to fulfill indispensable functions in light harvesting, protection
against photooxidation, and regulation of Photosystem II efficiency. The wealth of information accumulated in recent years
dealing with the closely related questions of carotenoid organization and functions are summarized in this chapter. In the
first section the distribution of carotenoids in the different pigment proteins is reported showing that each photosystem
subunit has its characteristic composition. The organization of the different xanthophylls within the antenna complexes is
discussed on the basis of recent structural and biochemical evidence. In the second section, advances in photophysical mechanisms
through which carotenoids perform their classical light harvesting and protective functions are discussed. In addition, particular
attention is given to discussion of the xanthophyll cycle which, in conjunction with the transthylakoid ΔpH1 down-regulates Photosystem II photochemical efficiency by non-radiative dissipation of energy in the light-harvesting complexes.
Down-regulation helps to keep PS II traps open, thereby helping to maintain electron transport and to protect the reaction
center from photoinhibition.
Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M−1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M−1 in the dark and only 300 M−1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′ATP was −42 kJ/mol in darkened intact chloroplasts, −46 kJ/mol in illuminated intact chloroplasts and −60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH4Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO2 reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO2 reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.
Organisms that rely on oxygenic photosynthesis are subject to the effects of photo-oxidative damage, which impairs the function of photosystem-II (PSII). This phenomenon has the potential to lower rates of photosynthesis and diminish plant growth. Experimental evidence shows that the steady-state oxidation–reduction level of the primary quinone acceptor (QA) of PSII is the parameter that controls photodamage under a variety of physiological and environmental conditions. When QA is reduced, excitation energy at PSII is dissipated via a charge-recombination reaction. Such non-assimilatory dissipation of excitation generates singlet oxygen that might act to covalently modify the photochemical reaction center chlorophyll. Under steady-state photosynthesis conditions, the reduction state of QA increases linearly with irradiance, thereby causing a correspondingly linear increase in the probability of photodamage. It is concluded that there is a low probability that photodamage will occur when QA is oxidized and excitation energy is utilized in electron transport, and a significantly higher probability when QA is reduced in the course of steady-state photosynthesis.
When unicellular algal cells are placed under anaerobic conditions, a large electrochemical gradient is built in darkness across the thylakoid membranes. We have estimated, in vivo, the amplitude of the Delta pH component of this transmembrane potential and shown that the Delta pH is twice as large as the Delta Psi. The amplitude of the Delta mu tildeH+ (approximately 110-140 mV) fits well with estimations based on the ATP/ADP ratio measured in green algae under the same conditions, suggesting that an equilibrium state is established across the thylakoid membrane. Therefore, under anaerobic dark incubation of algae, the electrochemical transmembrane potential is determined only by the cellular ATP content. The existence of this Delta mu tildeH+ is expected to result in a constitutive amount of activated CFo-CF1 ATPase, thereby facilitating ATP synthesis under low light intensity illumination. We report also on the effects of this dark-existing electrochemical gradient on the cytochrome b6f complex turnover kinetics. We show that they are largely slowed by the presence of this electrochemical transmembrane potential. The pH component is mainly responsible for the kinetic slowing down of cytochrome b6f complex turnover, despite the fact that electrogenicity is associated with the reactions taking place within this complex. Therefore, in vivo, owing to the low lumenal pH, the oxidation of plastoquinol at the Qo site is limiting the turnover of the cytochrome b6f complex in the presence of the Delta pH, while in its absence the oxidation rate of the b6 hemes becomes rate-limiting.
A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.
The observed levels of Delta G(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid proton motive force (pmf) equivalent to a Delta pH of approximately 2.5 units. If, as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than approximately 5. In contrast, we have presented evidence that the pH of the thylakoid lumen does not drop below pH approximately 5.8 [Kramer, D. M., Sacksteder, C. A., and Cruz, J. A. (1999) Photosynth. Res. 60, 151-163], leading us to propose that Delta psi can contribute to steady-state pmf. In this work, it is demonstrated, through assays on isolated thylakoids and computer simulations, that thylakoids can store a substantial fraction of pmf as Delta psi, provided that the activities of ions permeable to the thylakoid membrane in the chloroplast stromal compartment are relatively low and the buffering capacity (beta) for protons of the lumen is relatively high. Measurements of the light-induced electrochromic shift (ECS) confirm the ionic strength behavior of steady-state Delta psi in isolated, partially uncoupled thylakoids. Measurements of the ECS in intact plants illuminated for 65 s were consistent with low concentrations of permeable ions and approximately 50% storage of pmf as Delta psi. We propose that the plant cell, possibly at the level of the inner chloroplast envelope, can control the parsing of pmf into Delta psi and Delta pH by regulating the ionic strength and balance of the chloroplast. In addition, this work demonstrates that, under certain conditions, the kinetics of the light-induced ECS can be used to estimate the fractions of pmf stored as Delta psi and Delta pH both in vitro and in vivo.
Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.
The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.
Light-driven electron transport is coupled to ATP synthesis in chloroplasts. While the nature of the coupling and the structures of key components are now known, there has long been disagreement over pathways of electron transport. Recent results now put an old idea back on the agenda-cyclic electron transport around photosystem I.
The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.
Photosynthesis, stroma-pH, and internal K(+) and Cl(-) concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K(+), H(+), Cl(-)) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).Isolated intact chloroplasts are a Donnan system which accumulates cations (K(+) or added Tetraphenylphosphonium(+)) and excludes anions (Cl(-)) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium(+) distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K(+) or Na(+) and the release of protons all become very small. Light-induced K(+) uptake is not abolished by valinomycin suggesting that the K(+) uptake is not primarily active. Intact chloroplasts contain higher K(+) concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO(2) fixation at high K(+) concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO(2) fixation in intact chloroplasts.
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