Thylakoid Proton Motive Force Research Articles

Genetically manipulated chloroplast stromal phosphate levels alter photosynthetic efficiency.

The concentration of inorganic phosphate (Pi) in the chloroplast stroma must be maintained within narrow limits to sustain photosynthesis and to direct the partitioning of fixed carbon. However, it is unknown if these limits or the underlying contributions of different chloroplastic Pi transporters vary throughout the photoperiod or between chloroplasts in different leaf tissues. To address these questions, we applied live Pi imaging to Arabidopsis (Arabidopsis thaliana) wild-type plants and two loss-of-function transporter mutants: triose phosphate/phosphate translocator (tpt), phosphate transporter 2; 1 (pht2; 1), and tpt pht2; 1. Our analyses revealed that stromal Pi varies spatially and temporally, and that TPT and PHT2; 1 contribute to Pi import with overlapping tissue specificities. Further, the series of progressively diminished steady-state stromal Pi levels in these mutants provided the means to examine the effects of Pi on photosynthetic efficiency without imposing nutritional deprivation. ΦPSII and nonphotochemical quenching (NPQ) correlated with stromal Pi levels. However, the proton efflux activity of the ATP synthase (gH+) and the thylakoid proton motive force (pmf) were unaltered under growth conditions, but were suppressed transiently after a dark to light transition with return to wild-type levels within 2 minutes. These results argue against a simple substrate-level limitation of ATP synthase by depletion of stromal Pi, favoring more integrated regulatory models, which include rapid acclimation of thylakoid ATP synthase activity to reduced Pi levels.

H+ Transport by K+ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

In vivo regulation of thylakoid proton motive force in immature leaves.

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.

In vivo regulation of proton motive force during photosynthetic induction

Thylakoid proton motive force (pmf) plays a central role in photosynthesis. However, the regulation of pmf during photosynthetic induction is not well known. In the present study, in order to investigate the importance of cyclic electron flow (CEF) and chloroplastic ATP synthase in pmf formation during photosynthetic induction, we examined chlorophyll fluorescence, P700 signal, electrochromic shift signal for leaves of a low-light species Panax notoginseng and a high-light species Bletilla striata. During the initial induction phase at both low and high light, the activation of CEF associating with low proton conductivity of the chloroplastic ATP synthase (gH+) resulted in the rapid buildup of proton motive force (pmf) in both species. Furthermore, during photosynthetic induction at high light, the increase in gH+ in B. striata was accompanied with increases in linear electron flow (LEF) and CEF, leading to a stable pmf. Under high light, the high-light species B. striata, with much higher LEF and CEF than P. notoginseng, showed significantly lower pmf owing to the higher gH+. This result suggested that, under high light, CEF mainly contributed to ATP synthase in B. striata but favored lumenal acidification in P. notoginseng. Taking together, during photosynthetic induction, the coordination between CEF and the activity of chloroplastic ATP synthase finely regulated the pmf levels, optimizing photosynthesis and photoprotection.

In Silico Analysis of the Regulation of the Photosynthetic Electron Transport Chain in C3 Plants.

We present a new simulation model of the reactions in the photosynthetic electron transport chain of C3 species. We show that including recent insights about the regulation of the thylakoid proton motive force, ATP/NADPH balancing mechanisms (cyclic and noncyclic alternative electron transport), and regulation of Rubisco activity leads to emergent behaviors that may affect the operation and regulation of photosynthesis under different dynamic environmental conditions. The model was parameterized with experimental results in the literature, with a focus on Arabidopsis (Arabidopsis thaliana). A dataset was constructed from multiple sources, including measurements of steady-state and dynamic gas exchange, chlorophyll fluorescence, and absorbance spectroscopy under different light intensities and CO2, to test predictions of the model under different experimental conditions. Simulations suggested that there are strong interactions between cyclic and noncyclic alternative electron transport and that an excess capacity for alternative electron transport is required to ensure adequate redox state and lumen pH. Furthermore, the model predicted that, under specific conditions, reduction of ferredoxin by plastoquinol is possible after a rapid increase in light intensity. Further analysis also revealed that the relationship between ATP synthesis and proton motive force was highly regulated by the concentrations of ATP, ADP, and inorganic phosphate, and this facilitated an increase in nonphotochemical quenching and proton motive force under conditions where metabolism was limiting, such as low CO2, high light intensity, or combined high CO2 and high light intensity. The model may be used as an in silico platform for future research on the regulation of photosynthetic electron transport.

Hacking the thylakoid proton motive force for improved photosynthesis: modulating ion flux rates that control proton motive force partitioning into Δψ and ΔpH.

There is considerable interest in improving plant productivity by altering the dynamic responses of photosynthesis in tune with natural conditions. This is exemplified by the 'energy-dependent' form of non-photochemical quenching (qE), the formation and decay of which can be considerably slower than natural light fluctuations, limiting photochemical yield. In addition, we recently reported that rapidly fluctuating light can produce field recombination-induced photodamage (FRIP), where large spikes in electric field across the thylakoid membrane (Δψ) induce photosystem II recombination reactions that produce damaging singlet oxygen (1O2). Both qE and FRIP are directly linked to the thylakoid proton motive force (pmf), and in particular, the slow kinetics of partitioning pmf into its ΔpH and Δψ components. Using a series of computational simulations, we explored the possibility of 'hacking' pmf partitioning as a target for improving photosynthesis. Under a range of illumination conditions, increasing the rate of counter-ion fluxes across the thylakoid membrane should lead to more rapid dissipation of Δψ and formation of ΔpH. This would result in increased rates for the formation and decay of qE while resulting in a more rapid decline in the amplitudes of Δψ-spikes and decreasing 1O2 production. These results suggest that ion fluxes may be a viable target for plant breeding or engineering. However, these changes also induce transient, but substantial mismatches in the ATP : NADPH output ratio as well as in the osmotic balance between the lumen and stroma, either of which may explain why evolution has not already accelerated thylakoid ion fluxes. Overall, though the model is simplified, it recapitulates many of the responses seen in vivo, while spotlighting critical aspects of the complex interactions between pmf components and photosynthetic processes. By making the programme available, we hope to enable the community of photosynthesis researchers to further explore and test specific hypotheses.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented.Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions.

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection.

In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

Limitations to photosynthesis by proton motive force-induced photosystem II photodamage.

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.

MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network

Large-scale high-throughput plant phenotyping (sometimes called phenomics) is becoming increasingly important in plant biology and agriculture and is essential to cutting-edge plant breeding and management approaches needed to meet the food and fuel needs for the next century. Currently, the application of these approaches is severely limited by the availability of appropriate instrumentation and by the ability to communicate experimental protocols, results and analyses. To address these issues, we have developed a low-cost, yet sophisticated open-source scientific instrument designed to enable communities of researchers, plant breeders, educators, farmers and citizen scientists to collect high-quality field data on a large scale. The MultispeQ provides measurements in the field or laboratory of both, environmental conditions (light intensity and quality, temperature, humidity, CO2 levels, time and location) and useful plant phenotypes, including photosynthetic parameters—photosystem II quantum yield (ΦII), non-photochemical exciton quenching (NPQ), photosystem II photoinhibition, light-driven proton translocation and thylakoid proton motive force, regulation of the chloroplast ATP synthase and potentially many others—and leaf chlorophyll and other pigments. Plant phenotype data are transmitted from the MultispeQ to mobile devices, laptops or desktop computers together with key metadata that gets saved to the PhotosynQ platform (https://photosynq.org) and provides a suite of web-based tools for sharing, visualization, filtering, dissemination and analyses. We present validation experiments, comparing MultispeQ results with established platforms, and show that it can be usefully deployed in both laboratory and field settings. We present evidence that MultispeQ can be used by communities of researchers to rapidly measure, store and analyse multiple environmental and plant properties, allowing for deeper understanding of the complex interactions between plants and their environment.

Multi-level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance.

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ-subunit through the ferredoxin-thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild-type levels as irradiance was increased. This effect was caused by an altered redox state of the γ-subunit under low, but not high, light. The low light-specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non-photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin-thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.

Rethinking the existence of a steady-state Δψ component of the proton motive force across plant thylakoid membranes

Light-driven photosynthetic electron transport is coupled to the movement of protons from the chloroplast stroma to the thylakoid lumen. The resulting proton motive force that is generated is used to drive the conformational rotation of the transmembrane thylakoid ATPase enzyme which converts ADP (adenosine diphosphate) and Pi (inorganic phosphate) into ATP (adenosine triphosphate), the energy currency of the plant cell required for carbon fixation and other metabolic processes. According to Mitchell's chemiosmotic hypothesis, the proton motive force can be parsed into the transmembrane proton gradient (ΔpH) and the electric field gradient (Δψ), which are thermodynamically equivalent. In chloroplasts, the proton motive force has been suggested to be split almost equally between Δψ and ΔpH (Kramer et al., Photosynth Res 60:151-163, 1999). One of the central pieces of evidence for this theory is the existence of a steady-state electrochromic shift (ECS) absorption signal detected ~515nm in plant leaves during illumination. The interpretation of this signal is complicated, however, by a heavily overlapping absorption change ~535nm associated with the formation of photoprotective energy dissipation (qE) during illumination. In this study, we present new evidence that dissects the overlapping contributions of the ECS and qE-related absorption changes in wild-type Arabidopsis leaves using specific inhibitors of the ΔpH (nigericin) and Δψ (valinomycin) and separately using leaves of the Arabidopsis lut2npq1 mutant that lacks qE. In both cases, our data show that no steady-state ECS signal persists in the light longer than ~60s. The consequences of our observations for the suggesting parsing of steady-state thylakoid proton motive force between (ΔpH) and the electric field gradient (Δψ) are discussed.

The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf

Endogenous probes of light-induced transthylakoid proton motive force ( pmf), membrane potential (Δ ψ) and ΔpH were used in vivo to assess in Arabidopsis the lumen pH responses of regulatory components of photosynthesis. The accumulation of zeaxanthin and protonation of PsbS were found to have similar p K a values, but quite distinct Hill coefficients, a feature allowing high antenna efficiency at low pmf and fine adjustment at higher pmf. The onset of “energy-dependent’ exciton quenching ( q E) occurred at higher lumen pH than slowing of plastoquinol oxidation at the cytochrome b 6 f complex, presumably to prevent buildup of reduced electron carriers that can lead to photodamage. Quantitative comparison of intrinsic probes with the electrochromic shift signal in situ allowed quantitative estimates of pmf and lumen pH. Within a degree of uncertainly of ∼ 0.5 pH units, the lumen pH was estimated to range from ∼ 7.5 (under weak light at ambient CO 2) to ∼ 5.7 (under 50 ppm CO 2 and saturating light), consistent with a ‘moderate pH’ model, allowing antenna regulation but preventing acid-induced photodamage. The apparent p K a values for accumulation of zeaxanthin and PsbS protonation were found to be ∼ 6.8, with Hill coefficients of about 4 and 1 respectively. The apparent shift between in vitro violaxanthin deepoxidase protonation and zeaxanthin accumulation in vivo is explained by steady-state competition between zeaxanthin formation and its subsequent epoxidation by zeaxanthin epoxidase. In contrast to tobacco, Arabidopsis showed substantial variations in the fraction of pmf (0.1–0.7) stored as Δ ψ, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.