Chloroplast ion homeostasis - what do we know and where should we go?

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.

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria.

Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full "overcapacity" of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be "lost."

Optimization of ATP Synthase c-Rings for Oxygenic Photosynthesis.

The conversion of sunlight into useable cellular energy occurs via the proton-coupled electron transfer reactions of photosynthesis. Light is absorbed by photosynthetic pigments and transferred to photochemical reaction centers to initiate electron and proton transfer reactions to store energy in a redox gradient and an electrochemical proton gradient (proton motive force, pmf), composed of a concentration gradient (ΔpH) and an electric field (Δψ), which drives the synthesis of ATP through the thylakoid FoF1-ATP synthase. Although ATP synthase structure and function are conserved across biological kingdoms, the number of membrane-embedded ion-binding c subunits varies between organisms, ranging from 8 to 17, theoretically altering the H+/ATP ratio for different ATP synthase complexes, with profound implications for the bioenergetic processes of cellular metabolism. Of the known c-ring stoichiometries, photosynthetic c-rings are among the largest identified stoichiometries, and it has been proposed that decreasing the c-stoichiometry could increase the energy conversion efficiency of photosynthesis. Indeed, there is strong evidence that the high H+/ATP of the chloroplast ATP synthase results in a low ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio produced by photosynthetic linear electron flow, requiring secondary processes such as cyclic electron flow to support downstream metabolism. We hypothesize that the larger c subunit stoichiometry observed in photosynthetic ATP synthases was selected for because it allows the thylakoid to maintain pmf in a range where ATP synthesis is supported, but avoids excess Δψ and ΔpH, both of which can lead to production of reactive oxygen species and subsequent photodamage. Numerical kinetic simulations of the energetics of chloroplast photosynthetic reactions with altered c-ring size predicts the energy storage of pmf and its effects on the photochemical reaction centers strongly support this hypothesis, suggesting that, despite the low efficiency and suboptimal ATP/NADPH ratio, a high H+/ATP is favored to avoid photodamage. This has important implications for the evolution and regulation of photosynthesis as well as for synthetic biology efforts to alter photosynthetic efficiency by engineering the ATP synthase.

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.

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'.

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.

Dynamic Environmental Photosynthetic Imaging Reveals Emergent Phenotypes.

Understanding and improving the productivity and robustness of plant photosynthesis requires high-throughput phenotyping under environmental conditions that are relevant to the field. Here we demonstrate the dynamic environmental photosynthesis imager (DEPI), an experimental platform for integrated, continuous, and high-throughput measurements of photosynthetic parameters during plant growth under reproducible yet dynamic environmental conditions. Using parallel imagers obviates the need to move plants or sensors, reducing artifacts and allowing simultaneous measurement on large numbers of plants. As a result, DEPI can reveal phenotypes that are not evident under standard laboratory conditions but emerge under progressively more dynamic illumination. We show examples in mutants of Arabidopsis of such "emergent phenotypes" that are highly transient and heterogeneous, appearing in different leaves under different conditions and depending in complex ways on both environmental conditions and plant developmental age. These emergent phenotypes appear to be caused by a range of phenomena, suggesting that such previously unseen processes are critical for plant responses to dynamic environments.

Redox regulation of the antimycin A sensitive pathway of cyclic electron flow around photosystem I in higher plant thylakoids.

The chloroplast must regulate supply of reducing equivalents and ATP to meet rapid changes in downstream metabolic demands. Cyclic electron flow around photosystem I (CEF) is proposed to balance the ATP/NADPH budget by using reducing equivalents to drive plastoquinone reduction, leading to the generation of proton motive force and subsequent ATP synthesis. While high rates of CEF have been observed in vivo, isolated thylakoids show only very slow rates, suggesting that the activity of a key complex is lost or down-regulated upon isolation. We show that isolation of thylakoids while in the continuous presence of reduced thiol reductant dithiothreitol (DTT), but not oxidized DTT, maintains high CEF activity through an antimycin A sensitive ferredoxin:quinone reductase (FQR). Maintaining low concentrations (~2 mM) of reduced DTT while modulating the concentration of oxidized DTT leads to reversible activation/inactivation of CEF with an apparent midpoint potential of -306 mV (±10 mV) and n=2, consistent with redox modulation of a thiol/disulfide couple and thioredoxin-mediated regulation of the plastoquinone reductase involved in the antimycin A-sensitive pathway, possibly at the level of the PGRL1 protein. Based on proposed differences in regulatory modes, we propose that the FQR and NADPH:plastoquinone oxidoreductase (NDH) pathways for CEF are activated under different conditions and fulfill different roles in chloroplast energy balance.