Proton motive force partitioning links energy and redox balance to photoprotection and carbon gain

qH (hours to days): A sustained, PsbS-independent quenching mode requiring the plastid lipocalin LCNP and antagonized by SOQ1; recent work identifies LHCII trimers as sites associated with qH-type dissipation (Malnoë, 2018, 2018; Bru et al., 2022).

The system performs well when ΔpH kinetics align with the response windows of the effectors:

Seconds (sunfleck onset): ΔpH rises rapidly; PsbS protonation enables fast qE engagement, and photosynthetic control at b6f can restrict electron delivery to PSI if sinks lag.

Minutes (continued high light or repeated sunflecks): Sustained ΔpH supports VDE activity and zeaxanthin accumulation, increasing quenching magnitude and persistence (qZ-like behavior).

Hours to days (chronic stress): qI and/or qH can dominate; these states protect but must be resolved to avoid long-lasting suppression of photochemical yield after conditions improve (Roach and Krieger-Liszkay, 2014; Malnoë, 2018).

Therefore, upstream partitioning controls—ATP synthase g(H+), KEA3-dependent ΔpH relaxation after downshifts, and Δψ-relieving anion pathways (VCCN1/CLCe)—indirectly determine when PsbS and VDE engage and how long quenching persists. This explains why thylakoid ion transport mutants can show large changes in NPQ amplitude and relaxation even when antenna proteins are unchanged (Davis et al., 2017; Armbruster et al., 2016; Dukic et al., 2019, 2022).

Everything upstream in the proton circuit—pmf production, its ΔpH:Δψ partitioning, and pH-sensitive switches—exists to stabilize carbon fixation and the broader metabolic network under real, fluctuating environments (Yamori et al., 2016; Gollan et al., 2023; Wu et al., 2024; Moland et al., 2025). In this section, lumen acidification and stromal alkalization are connected to activation of the Calvin–Benson–Bassham (CBB) cycle, to ATP/NADPH balancing via cyclic or alternative electron routes and metabolite shuttles, in addition to the dynamic control of assimilation during sun- and shade flecks (Davis et al., 2017).

Light-driven proton pumping acidifies the lumen while alkalinizing the stroma, and this pH reset is not ancillary; it is required to bring several CBB enzymes into their high-activity regime and to coordinate them with the light reactions (Walker et al., 1986; Wang and Portis, 2006; Gregory et al., 2024). In vivo analyses and synthetic views converge on the point that stromal alkalization, with concomitant Mg²+ release into the stroma, promotes activation of FBPase, SBPase, and PRK, facilitates reductive activation by the thioredoxin system so that carbon-fixation capacity tracks light availability (Okegawa and Motohashi, 2015). Rubisco activase (RCA) links this protonic state to rubisco engagement, because its ATP/ADP- and redox-sensitive regulation makes it responsive to the chloroplast energy/redox state established by pmf and electron transport. Mechanistic and structural evidence further supports a model in which redox control tunes RCA nucleotide sensitivity, providing a molecular basis for light-dependent Rubisco activation (Wang and Portis, 2006). The proton circuit, therefore, does not merely supply ATP; it reconfigures the stromal microenvironment—pH, Mg²+, redox—so that the CBB cycle runs at the “right” set point for the current light and can be quickly dialed back when light fades (Werdan et al., 1975).

The “switch-on” logic is incomplete if treated only as pH/Mg²+/redox. ATP synthase requires Pi as a substrate, and its throughput is constrained by the return of ADP and Pi from stromal metabolism. When Pi availability is low (e.g., because Pi is sequestered in phosphorylated intermediates, export is restricted, or TPU limitation develops), proton efflux through ATP synthase can be throttled even if pmf is high, strengthening ΔpH-dependent regulation and slowing relaxation after downshifts (Kiirats et al., 2009; McClain et al., 2023). In this view, stromal alkalinization and enzyme activation set the 3.62.6 Contextual determinants of NPQ kinetics and amplitude for carbon fixation, whereas adenylate/Pi supply and sink turnover set the realized flux and strongly influence the kinetics of recovery after fluctuations.

By stoichiometry, LEF (Linear Electron Flow) provides ~1.3 ATP per NADPH, whereas C3 carbon fixation plus photorespiration often demands more ATP per NADPH, especially in warm, variable light (Cruz et al., 2001). This mismatch necessitates pmf-boosting and ATP-biasing mechanisms—most notably CEF around PSI and NDH-dependent routes—which add protons, and thus ATP potential, without net NADPH production (Cruz et al., 2001; Shikanai, 2007; Buchanan, 2017). A recent review on “alternative power lines” argues that multiple auxiliary routes—CEF (Cyclic electron flow) via PGR5/PGRL1, NDH, and photorespiratory or respiratory couplings—dynamically rebalance ATP/NADPH to sustain carbon metabolism when LEF alone would undersupply ATP (Davis et al., 2017; Kedem et al., 2021). In C4 leaves, where bundle-sheath ATP demand is higher due to the CO2-concentrating cycle, NDH-mediated CEF has been quantified in vivo as the dominant PSI electron route in bundle-sheath chloroplasts, underscoring the centrality of proton-pumping routes for sustaining assimilation in these tissues (Ermakova et al., 2019). When reductant accumulates (high NADPH/low ATP), chloroplasts export reducing equivalents via the NADP-MDH “malate valve,” handing off redox to the cytosol or mitochondria and indirectly helping recycle NADP+ for PSI while supporting ATP production outside the chloroplast; this shuttle is repeatedly highlighted as a key buffer that couples the proton circuit to downstream metabolism (Figure 3) (Scheibe, 2004; Selinski et al., 2014; Selinski and Scheibe, 2019).

In natural light, assimilation rarely reaches steady state, and the timing of ΔpH formation and relaxation—set by pumps, the ATP synthase “valve,” and counter-ion routes—gates both protection and productivity on sub-minute scales (Werdan et al., 1975; Michelet et al., 2013; Savage et al., 2016; Walker et al., 2020). At sunfleck onset (low to high light), a rapid ΔpH spike triggers qE and photosynthetic control at b6f, preventing PSI over-reduction while Rubisco and RCA catch up; because PSI lacks a rapid repair cycle, this prioritization averts damage that would depress CO2 uptake for days. At shadefleck onset (high to low light), the rate of ΔpH relaxation limits recovery of PSII quantum yield and thus CO2 assimilation; KEA3, a thylakoid K+/H+ antiporter whose C-terminus senses chloroplast energy state, accelerates qE and ΔpH relaxation and improves post-shadefleck efficiency. Under chronic fluctuation, insufficient biasing of ΔpH at upshifts—due to weak anion or cation counter-fluxes—leads to PSI photoinhibition, slow recovery, and assimilation constraints that far outlast the transient; recent syntheses and experiments place b6f “photosynthetic control” at the center of this protective logic (Werdan et al., 1975; Avenson et al., 2005; Hisabori et al., 2013; Davis et al., 2017; Foyer and Noctor, 2020). Faster assimilation transients are observed when sink strength rises; for example, elevated CO2 lowers PSI over-reduction during light transitions by pulling electrons into the CBB cycle more rapidly (Kono and Terashima, 2016).A critical addition is that Pi recycling can be rate-limiting in both directions. During rapid induction, Pi availability influences ATP synthase throughput and therefore the pace at which ATP supply rises to support CBB activation and RuBP regeneration (Kiirats et al., 2009).

During recovery, Pi sequestration in phosphorylated intermediates and limitations in export/end-product use can slow ATP synthase flux and prolong ΔpH, delaying NPQ relaxation and suppressing assimilation even when incident light has decreased (McClain and Sharkey, 2019; McClain et al., 2023). These effects help explain why assimilation transients often reflect combined “biophysical timing” (ΔpH dynamics) and “biochemical timing” (Pi return and sink engagement), and why interpreting NPQ kinetics as a direct proxy for ΔpH timing can be misleading without concurrent assessment of pmf partitioning and Pi/export status.

ΔpH is the shared trigger for qE, xanthophyll cycling, and b6f control, the same levers that set pmf partitioning, also tune CO2-uptake dynamics (Michelberger et al., 2025). Lower ATP synthase conductance (g(H+)) preserves ΔpH for protection but can transiently restrict ΦPSII and ATP supply, whereas higher conductance speeds ATP delivery and ΔpH collapse but risks PSI if electron sinks lag; the optimal g (H+) depends on the light-fluctuation pattern and downstream sink capacity (Michelet et al., 2013; Johnson et al., 2014; Davis et al., 2017). KEA3 modulates ΔpH relaxation in response to stromal energy state (ATP/ADP, pH) and thereby helps avoid “over-holding” qE after shadeflecks, reducing unnecessary losses in ΦPSII and boosting assimilation (Armbruster et al., 2016; Wang and Shikanai, 2019). Rapid Δψ relief through VCCN1 biases pmf to ΔpH at sunflecks, strengthening protective throttling at b6f and preserving PSI, which maintains assimilation capacity over hours to days. The overarching design principle is that the proton circuit must be tuned not for maximal ΔpH or maximal ATP alone, but for the right ΔpH at the right time, preserving PSI while minimizing unnecessary qE carry-over that would tax ΦPSII and CO2 uptake (Wilson et al., 2021; Hagino et al., 2022).

Downstream of the CBB cycle, the triose-phosphate/phosphate translocator (TPT) exchanges triose-P for inorganic phosphate (Pi), thereby coupling chloroplast export to Pi recycling for ATP synthase. Under high sugar build-up or low Pi return, leaves can enter triose phosphate utilization (TPU) limitation, in which assimilation is capped by end-product processing or export rather than by Rubisco activity or RuBP regeneration. Recent physiological and modeling studies treat TPU as a systems-level property of the source–sink network that becomes especially prominent under specific combinations of CO2, temperature, and sink strength, rather than as a simple, static biochemical constant (Quick and Schaffer, 2017; McClain and Sharkey, 2019; Rogers et al., 2021). Because ATP synthase requires Pi as substrate, Pi availability—and thus TPT-mediated return—feeds back onto ATP production from pmf, providing another route by which carbon-export status influences how proton-motive energy is cashed out (Cruz et al., 2001; Golding and Johnson, 2003).

Beyond transport stoichiometry, source–sink status is coordinated by broader regulatory networks that couple carbohydrate availability, phloem transport capacity, and stress responses. While a full treatment is beyond the scope of this review, the central implication for a proton-circuit framework is that carbon-status signaling and sink strength can modulate the same proton-circuit variables that determine ΔpH timing, including g(H+), the persistence of ΔpH, and the degree to which photoprotective responses are maintained across repeated fluctuations (Savage et al., 2016; Quick and Schaffer, 2017). Likewise, photorespiration reshapes ATP demand and carbon flux partitioning, altering when ATP supply versus protection becomes the dominant constraint. These couplings motivate interpreting “proton-centric” diagnostics (ECS-based pmf/partitioning and g(H+), NPQ, P700) alongside carbon-status indicators (gas exchange, ACi/TPU signatures, and—where feasible—pigment state and Pi/nutrient context) to attribute limitations correctly in intact leaves (Walker et al., 2020; Rogers et al., 2021; McClain et al., 2023).

Plant thylakoids form a connected membrane network organized into appressed grana stacks and unappressed stroma lamellae, with grana margins providing curved transition regions between the two. This architecture is associated with lateral segregation of photosynthetic complexes: PSII–LHCII is enriched in grana cores, whereas PSI and chloroplast ATP synthase are largely excluded from grana cores and enriched in stroma-exposed membranes; cytochrome b 6 f is distributed across domains and has frequently been reported to show enrichment toward grana margins (Daum and Kühlbrandt, 2011; Anderson et al., 2012; Kirchhoff, 2013, 2014).

A key point for proton dynamics is that the lumen is topologically continuous, so lateral equilibration of lumen protons is possible in principle. However, continuity does not imply instantaneous equilibration: electron-tomography and diffusion-based frameworks emphasize that narrow junctions at margins, tortuous geometry, and crowding can slow effective diffusion and create conditions where local proton chemistry differs transiently across domains (Daum and Kühlbrandt, 2011; Kirchhoff, 2014; Kirchhoff et al., 2008).

The simplest physical reason to expect heterogeneity is spatial separation of proton sources and sinks. PSII and cytochrome b6 f deposit protons into the lumen, while ATP synthase provides the dominant proton return (“valve”), and these components are not co-localized (Kirchhoff, 2013, 2014). During light transients, this source–sink separation can generate lateral gradients in proton concentration and local pmf partitioning (ΔpH vs Δψ), especially when counter-ion pathways that dissipate Δψ are locally limiting. In this view, domain-level heterogeneity is expected to be most pronounced on seconds-to-minutes timescales where pumping/efflux rates change quickly (Tikhonov, 2012; Davis et al., 2017).

A second contributor is the local balance of charge compensation. Experiments and theory show that ionic strength and counter-ion movements can change how pmf is expressed as Δψ versus ΔpH, even when total pmf is similar (Cruz et al., 2001; Davis et al., 2017). While most measurements of Δψ/ΔpH are spatially averaged, the same logic implies that regional differences in ion permeability (anion influx and cation efflux pathways) could yield domain-dependent partitioning during transients (Davis et al., 2017).

Thylakoid organization is not static. State transitions remodel antenna connectivity and excitation distribution on minute timescales through STN7-dependent LHCII phosphorylation, changing the coupling between PSII and PSI and altering the spatial pattern of excitation pressure (Minagawa, 2011; Grieco et al., 2012; Mekala et al., 2015). Recent work also indicates additional STN7-linked regulatory behaviors beyond the simplest “LHCII moves” cartoon, reinforcing that multiple remodeling routes can coexist (Węgrzyn et al., 2022). Because excitation distribution helps set where electron flow (and thus proton deposition) is strongest, these reorganizations are expected to influence proton budgets indirectly, even if pmf itself remains globally dominated by ΔpH. Modeling frameworks for state transitions and membrane interactions provide useful constraints on how these reorganizations can change effective connectivity and diffusion geometry (Wood and Johnson, 2020).

In parallel, lumen swelling and osmotic responses accompany illumination and lumen acidification. Water flux following ion movements changes lumen dimensions and can therefore change effective diffusion distances and buffering capacity, implying that proton equilibration kinetics are themselves dynamically modulated by the light state (Tikhonov, 2012; Kaňa et al., 2016).

ATP synthase is concentrated in stroma-exposed membranes and is largely excluded from grana cores, establishing spatially localized proton efflux zones that are separated from the strongest PSII proton sources in grana. This geometry alone makes transient lateral ΔpH gradients plausible unless fast charge-compensating ion fluxes minimize local Δψ buildup (Kirchhoff, 2013, 2014; Davis et al., 2017).

For ion pathways that reshape partitioning, molecular identity is now strong, but sub-domain localization is still a limiting uncertainty for spatial models. VCCN1 has been structurally resolved as a bestrophin-like homopentamer, but the in vivo distribution across grana margins versus stroma lamellae is not yet firmly mapped; because VCCN1 contributes to Δψ relief and supports rapid ΔpH expression during illumination, its placement will strongly influence whether ΔpH rises quickly at the relevant sites during upshifts (Herdean et al., 2016b; Dukic et al., 2019; Hagino et al., 2022). CLCe is genetically linked to chloroplast ion homeostasis and to regulatory responses that include LHCII phosphorylation behavior, but its spatial deployment across domains also remains uncertain, again limiting predictive modeling of Δψ dissipation in space (Herdean et al., 2016a; Dukic et al., 2022). KEA3 is best treated as a ΔpH relaxation module after downshifts; mapping where KEA3 is enriched (margins vs lamellae vs broader distribution) is essential for predicting where ΔpH relaxation initiates and how quickly it propagates (Armbruster et al., 2016; Wang and Shikanai, 2019).

A conservative, testable working hypothesis is that grana margins act as regulatory “exchange zones” because they juxtapose (i) a transition region between grana and lamella membranes, (ii) cytochrome b6 f distribution that often shows margin bias, and (iii) proximity to ATP synthase-rich stroma-exposed regions. If Δψ-relieving pathways (e.g., VCCN1/CLCe) are also enriched at margins, this would favor rapid ΔpH expression during upshifts and rapid engagement of ΔpH-dependent controls. This remains a hypothesis until localization and local-pH readouts are directly measured (Kirchhoff, 2013, 2014; Davis et al., 2017; Hagino et al., 2022).

Finally, the TPK3 localization debate provides a cautionary precedent: putative thylakoid channels should not be treated as established spatial players without convergent localization and functional evidence (Carraretto et al., 2013; Höhner et al., 2019).

At seconds-to-minutes timescales, leaf photosynthesis is well described by a five-node circuit organized into two nested feedback loops (Kiirats et al., 2009; Hohmann-Marriott and Blankenship, 2011). Proton-pumping modules, PSII, cytochrome b6f, and CEF, raise the total pmf. Counter-ion pathways—notably KEA3 on the K+/H+ exchange side and VCCN1/CLCe on the anion side—reshape that pmf toward its chemical component (ΔpH) by relieving the electrical component (Δψ) (Dukic et al., 2022; Hagino et al., 2022). ATP synthase then spends pmf as ATP, and its effective proton conductance, gH+, sets the rate at which ΔpH collapses after a change in light or metabolic demand. pH-sensitive effectors read the ΔpH signal and throttle photochemistry in real time: PsbS mediates rapid, reversible qE; the VDE/ZE (violaxanthin de-epoxidase/zeaxanthin epoxidase) xanthophyll cycle establishes the longer-lived, zeaxanthin-dependent quenching state; and ΔpH-gated “photosynthetic control” at b6f slows plastoquinol oxidation when sinks lag. Carbon metabolism (the Calvin–Benson–Bassham enzymes together with Rubisco activase and export/transport steps) draws down electrons and ATP and thereby feeds back on the upstream redox and energy state that determines how much pmf is built and how it is partitioned. In compact terms, the fast protective loop raises ΔpH, engages PsbS and b6f control, and keeps PSI oxidized, whereas the productivity loop converts pmf into ATP, activates the CBB cycle, strengthens sinks, and relieves upstream over-reduction (Werdan et al., 1975; Zu Tittingdorf et al., 2004; Dall’Osto et al., 2012; Carmo-Silva and Salvucci, 2013; Hisabori et al., 2013; Wilson et al., 2021; Carbonera et al., 2022; Juhaszova et al., 2022).

The operational objective of the chloroplast proton-control network is conserved across genotypes and environments: to generate the right ΔpH at the right time. Too little ΔpH during a light upshift delays engagement of qE and cytochrome b6f–dependent photosynthetic control, increasing the risk of acceptor-side over-reduction at PSI and long-lived photoinhibition; too much ΔpH—or ΔpH that relaxes too slowly after a downshift—carries unnecessary NPQ into shade and suppresses ΦPSII and CO2 uptake (Zivcak et al., 2015; Allahverdiyeva et al., 2015; Tikhonov, 2018; Bethmann et al., 2019; Didaran et al., 2024). The same levers that shape pmf therefore set where the leaf sits on the photoprotection–productivity trade-off: anion flux pathways (e.g., VCCN1/CLCe) dissipate Δψ and thereby favor rapid ΔpH formation during light increases; KEA3 together with g(H+) sets the pace of ΔpH relaxation after transitions to lower light; and the balance between linear and cyclic electron flow determines how much pmf is generated in the first place. Because thylakoid architecture can impose spatial heterogeneity—microdomain ΔpH near b6f/PSII versus ATP synthase–rich regions—local kinetics can shape how quickly protection is engaged and how quickly antennae reopen (Armbruster et al., 2016; Wang and Shikanai, 2019). Viewed this way, performance is a timing problem rather than a maximization problem: protection benefits from a prompt ΔpH pulse during light upshifts, whereas productivity benefits from timely ΔpH relaxation during downshifts so that quenching does not outlast the need for it (Werdan et al., 1975; Wilson et al., 2021).

The operating-regime map that follows turns the proton-centric model into an experimenter’s field guide. For each common context in intact leaves, it links how the pmf is partitioned between ΔpH and Δψ, which controllers and effectors are expected to respond (KEA3, VCCN1/CLCe, ATP-synthase gH+, PsbS-mediated qE, and ΔpH-gated control at cytochrome b6f), and what carbon outcomes should result, including assimilation rate A, ΦPSII, and the redox status and safety of PSI (Wang and Shikanai, 2019; Bru et al., 2021). The map is organized around regimes that recur in practice: seconds-scale sunflecks and shadeflecks; dark-to-light induction; periods of high sink strength such as elevated CO2; counter-ion–limited genotypes (vccn1, kea3, clce); situations with high ATP bias in photorespiring C3 leaves; TPU/Pi limitation mediated by triose-phosphate export and phosphate return; state-transition–driven antenna remodeling; low-temperature, high-light conditions that permit qH; methodological pitfalls in pmf partitioning such as Δψ misassignment; and proton geography in which microdomain ΔpH governs local kinetics.

For each light condition, Table 3 specifies what balance of ΔpH and Δψ to expect, which protective and regulatory switches should engage, how net CO2 assimilation (A) is likely to behave, and which measurements resolve remaining ambiguities. The minimal, comparable measurement set includes ECSt as a pmf proxy; the ΔpH fraction from multi-wavelength ECS; gH+ from DIRK analysis; NPQ and P700 kinetics; ACi curves; xanthophyll de-epoxidation state; and Pi/TPT status where TPU is suspected (Table 3).

An operating-regime map is most informative when anchored to realistic pH set points that translate directly into the chemical component of the pmf. In intact leaves, illumination typically drives the stroma toward alkaline values near pH 8 while acidifying the lumen. Under strong light the lumen approaches roughly pH 5.5, whereas moderate light yields values nearer pH 6.0, and both compartments relax toward ~7.4 during light-to-dark transitions. Converting these pairs with (2.303 RT/F) ΔpH gives a chemical term of about 148 mV for high light (ΔpH=2.5), 118 mV for low–moderate light (ΔpH=2.0), and essentially zero in darkness (Werdan et al., 1975; Herdean et al., 2016; Hagino et al., 2022). These lumen and stromal pH dynamics and provide a quantitative backdrop for the pmf bars in Figure 4, which stack the computed ΔpH contribution above an electrical base representing Δψ. In vivo, Δψ tends to be smaller in bright light, somewhat larger at moderate light, and negligible after darkening, because ion movements dissipate the field as conditions change (Cruz et al., 2001; Wilson et al., 2021) (Figure 4).