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

When g(H+) is low, pmf (often ΔpH) can rise higher and persist longer, strengthening qE and photosynthetic control—but prolonged low conductance can constrain ATP delivery and delay recovery of photosynthetic yield after light decreases (Kiirats et al., 2009; Davis et al., 2017).

Crucially, g(H+) is not a “leaf constant”: it responds to redox regulation and also to metabolic state (ADP, Pi availability, and stromal demand), creating a feedback loop in which carbon metabolism can modulate how rapidly pmf is cashed out as ATP (Kiirats et al., 2009; Correa Galvis et al., 2020). Recent mechanistic work in algae further emphasizes that regulatory tuning of ATP synthase can differ across lifestyles and environmental regimes, reinforcing that ATP synthase control is a legitimate regulatory lever rather than a passive boundary condition (Rühle et al, 2024).

Because Δψ and ΔpH sum to the same pmf, ion movements that dissipate Δψ can increase the fraction of pmf expressed as ΔpH, allowing faster and deeper lumen acidification for a given net proton deposition. This “Δψ-relief to ΔpH-expression” logic underlies the importance of thylakoid anion and cation pathways for rapid photoprotection during light increases (Cruz et al., 2001; Davis et al., 2017).

The bestrophin-like channel VCCN1 provides anion influx that dissipates Δψ and thereby supports rapid ΔpH formation and timely engagement of qE and photosynthetic control during illumination (Duan et al., 2016; Herdean et al., 2016b; Dukic et al., 2019). Structural work has resolved VCCN1 as a bestrophin-like homopentamer and provides constraints for future kinetic models of its gating and conductance behavior in vivo (Hagino et al., 2022). In parallel, the CLC-family transporter/channel CLCe contributes to chloroplast ion homeostasis and influences photosynthetic regulation; genetic analyses link CLCe to ATP availability for LHCII phosphorylation and to acclimatory responses under particular light regimes (Figure 2) (Herdean et al., 2016a; Dukic et al., 2022).

In contrast to VCCN1/CLCe, the thylakoid K+/H+ antiporter KEA3 predominantly functions as a ΔpH relaxer after transitions to lower light by exchanging stromal K+ for luminal H+, accelerating qE relaxation and improving recovery of photochemical efficiency when strong lumen acidification is no longer required (Armbruster et al., 2014, 2016; Wang and Shikanai, 2019). Mechanistic studies further indicate that KEA3 regulation is coupled to chloroplast energy state, reinforcing that ion transport is integrated with metabolism and ATP synthase control rather than operating independently (Figure 2) (Armbruster et al., 2016; Correa Galvis et al., 2020; Uflewski et al., 2021).

Single- and multi-mutant studies (vccn1, clce, kea3 combinations) support the systems-level view that plants tune Δψ dissipation and ΔpH formation/relaxation to balance rapid protection with efficient recovery under fluctuating light (Dukic et al., 2019, 2022; Gollan et al., 2023). Phenotypes under fluctuating regimes are particularly informative because they report on timing: insufficient ΔpH expression during upshifts compromises protection, whereas excessively persistent ΔpH after downshifts sustains unnecessary quenching.

TPK3 is treated as a localization cautionary case, not a settled thylakoid component. TPK3 was initially reported as thylakoid-associated with photosynthetic consequences (Carraretto et al., 2013), but later work found photosynthesis largely unaffected by loss of TPK3 function and supports a vacuolar (tonoplast) role, illustrating why transporter identity and localization must be treated conservatively in “transportome” schematics (Höhner et al., 2019).

Proton deposition rises immediately. Δψ can spike rapidly at first, but effective Δψ dissipation through anion influx (e.g., VCCN1/CLCe-dependent pathways) enables a rapid rise in ΔpH, engaging qE and ΔpH-dependent photosynthetic control at cytochrome b6f before PSI acceptor-side limitation becomes severe. If g(H+) is low enough (or transiently restricted), ΔpH rises higher and protection engages more strongly; if g(H+) is very high and/or Δψ dissipation is weak, ΔpH expression is delayed and PSI becomes more vulnerable (Cruz et al., 2001; Davis et al., 2017; Herdean et al., 2016b; Dukic et al., 2019).

Proton deposition falls. Recovery requires timely relaxation of ΔpH so that qE does not persist longer than necessary. KEA3 accelerates ΔpH decay after downshifts, and ATP synthase conductance (g(H+)) also contributes strongly to the decay constant; together, these controls set the recovery kinetics of photochemical yield. Notably, NPQ relaxation kinetics also depend on pigment-state dynamics (e.g., zeaxanthin epoxidation) (Armbruster et al., 2016; Wang and Shikanai, 2019).

Under repeated fluctuations, loss of coordinated “shaper” function can lead to mis-timed protection (ΔpH forms too slowly at upshifts) and/or mis-timed recovery (ΔpH relaxes too slowly at downshifts), increasing PSI photoinhibition risk and depressing integrated carbon gain—phenotypes reported in combined thylakoid ion transport mutants under fluctuating light (Dukic et al., 2019; Gollan et al., 2023).

In the sections “upshift/downshift” logic to link pmf partitioning to rapid photoprotective engagement and recovery, and NPQ (especially qE) is often treated as a convenient kinetic readout of how rapidly lumen acidification is expressed and relaxed. However, NPQ is ΔpH-gated rather than ΔpH-determined: for an equivalent lumen pH trajectory, the sensitivity (pH threshold), amplitude, and relaxation of NPQ can differ substantially with genotype, pigment composition, and environmental or metabolic history. Conversely, differences in NPQ kinetics cannot be interpreted uniquely as differences in ΔpH timing unless pmf/partitioning (e.g., ECS-derived ΔpH fraction and g(H+)) and, where relevant, pigment state are assessed in parallel. These dependencies are especially important under fluctuating light, where “pre-conditioning” (previous sunflecks, stromal redox state, and temperature) can change the apparent speed and magnitude of NPQ independently of the underlying proton-circuit dynamics.

Table 2 summarizes major factors that shift NPQ induction and relaxation in intact leaves, providing a practical guide for interpreting NPQ transients without implicitly assuming a uniform activation model (Table 2).

At the level of first principles, pmf (ΔH+) equals the sum of Δψ and the chemical term (2.303 RT/F)· ΔpH. Conceptually, Δψ is shaped primarily by VCCN1, CLCe, and cation fluxes, whereas the ΔpH term is set by the balance between proton pumps and ATPase-mediated efflux, modulated by KEA3. Operationally, pumps (LEF/CEF) set the supply of pmf, ATP synthase (g(H+)) sets how fast pmf is spent as ATP, and VCCN1/CLCe/KEA3 (plus other ion fluxes) shape how much of that pmf is expressed as lumen acidification and how quickly it relaxes. This partitioning tunes the engagement of ΔpH-dependent photoprotective responses (qE and photosynthetic control at b6f) and therefore the stability of PSI under fluctuating light (Cruz et al., 2001; Davis et al., 2017; Armbruster et al., 2016; Gollan et al., 2023) (Figure 3).

Partitioning of pmf into ΔpH versus Δψ matters because ΔpH directly sets lumen pH, which serves as a chemical signal that engages multiple photoprotective “switches” on seconds-to-minutes timescales. These switches act on two complementary control axes: (i) excitation dissipation upstream of charge separation (NPQ, especially qE and qZ) and (ii) electron-transfer throttling within the chain (ΔpH-dependent photosynthetic control at cytochrome b6f). Together, they reduce excitation pressure on PSII and restrict electron delivery to PSI when stromal sinks lag, lowering the likelihood of ROS formation during rapid light changes (Papageorgiou and Govindjee, 2014; Davis et al., 2017; Degen and Johnson, 2024).

Lumen acidification should be treated as a gate rather than a single-input “driver.” For a given ΔpH trajectory, the realized amplitude and kinetics of quenching depend strongly on genotype (e.g., PsbS abundance/variants), pigment composition and xanthophyll pool state, antenna organization and membrane/lipid environment, and the recent light and metabolic history that sets stromal redox and substrate availability (Li et al., 2000, 2002; Correa-Galvis et al., 2016; Demmig-Adams et al., 2020). Moreover, fast regulatory layers are not strictly proton-dependent—such as protein phosphorylation/state transitions and redox signaling—which can reshape excitation distribution and antenna connectivity, thereby interacting with ΔpH-gated switches (Johnson and Ruban, 2014; Rochaix, 2014; Minagawa, 2011). These dependencies are particularly important when interpreting NPQ kinetics under fluctuating light, where “pre-conditioning” can shift apparent induction/relaxation even if pmf is similar.

The dominant fast NPQ component in plants, qE, is triggered by lumen acidification and is strongly dependent on the small thylakoid protein PsbS (Li et al., 2000, 2002). PsbS is a member of the light-harvesting complex superfamily but does not act as the quencher itself, because it does not bind a stable set of antenna pigments in the way LHC proteins do. Instead, PsbS functions as a ΔpH-responsive regulatory module that alters the interaction network and conformational landscape of PSII antenna proteins, increasing the probability of forming quenched antenna states when lumen pH drops (Papageorgiou and Govindjee, 2014; Correa-Galvis et al., 2016).

Mechanistically, qE activation involves protonation of conserved lumen-exposed acidic residues in PsbS (often described as two key glutamates in Arabidopsis; residue numbering can vary depending on how the N-terminus is defined and processed). Structural and residue-level analyses support the view that protonation shifts PsbS toward conformations/interactions that favor antenna reorganization and quenching competence, rather than creating a pigment-localized quenching site within PsbS itself (Fan et al., 2015; Liguori et al., 2019). Residue-by-residue studies further suggest that additional local sequence features (e.g., lumen loop segments and hydrophobic patches on transmembrane helices) tune sensitivity and kinetics, which is important for explaining why qE can differ in amplitude and relaxation across genotypes and environments (Liguori et al., 2019; Correa-Galvis et al., 2016).

Because PsbS lacks pigments, the dominant quenching sites are thought to reside in LHCII and/or minor antenna complexes, and multiple quenching locations likely contribute depending on pigment composition and membrane state (Mozzo et al., 2008; Papageorgiou and Govindjee, 2014). Zeaxanthin is not a strict prerequisite for the fastest qE onset in all contexts, but it can substantially enhance and stabilize quenching states and alter relaxation, linking qE mechanistically to the xanthophyll cycle (Li et al., 2002; Demmig-Adams et al., 2020).

The npq4 phenotype demonstrates that loss of PsbS strongly suppresses qE and changes the dynamics of energy dissipation during light transitions (Li et al., 2000, 2002). However, PSI protection should not be attributed to PsbS/qE alone: electron-transfer control (e.g., PGR5-dependent regulation and ΔpH-dependent photosynthetic control at b6f) can dominate PSI safety under many fluctuating-light regimes, so claims that “PsbS loss causes PSI photoinhibition” must be framed as conditional and supported by direct data (Tikkanen et al., 2015; Kono and Terashima, 2016; Kono et al, 2022). Conversely, engineering studies show that accelerating NPQ relaxation (often involving PsbS plus additional components) can improve carbon gain under fluctuating light, whereas overly persistent quenching can reduce yield when the light regime does not demand sustained dissipation (Kromdijk et al., 2016; Kromdijk and Walter, 2023).

The xanthophyll cycle couples lumen acidification to reversible changes in carotenoid composition that modulate both the magnitude and persistence of NPQ. Under acidic lumen pH, violaxanthin de-epoxidase (VDE) is activated on the lumenal side and uses ascorbate to convert violaxanthin to antheraxanthin and zeaxanthin, enriching the antenna with pigments that stabilize quenching states and improve photoprotection (Demmig-Adams et al., 2020). Importantly, VDE activity is not only pH-dependent but also lipid-dependent; evidence and models support recruitment/activation in MGDG-rich membrane environments, making local lipid organization a plausible contributor to heterogeneity in quenching competence (Goss and Latowski, 2020).

During lower light or recovery phases, zeaxanthin epoxidase (ZE) reconverts zeaxanthin toward violaxanthin on the stromal side, promoting relaxation. Under photoinhibitory stress, ZE abundance or activity can decrease, prolonging zeaxanthin retention and thereby extending the lifetime of quenching states—an effect consistent with ZE degradation under PSII photoinhibition conditions (Bethmann et al., 2019; Demmig-Adams et al., 2020).

PsbS provides fast ΔpH-gated switching (seconds) that defines the rapid onset and reversibility of qE.

Zeaxanthin modulates amplitude and persistence (minutes), contributing to qZ-like behavior and slower relaxation when zeaxanthin remains elevated (Li et al., 2002; Demmig-Adams et al., 2020).

Thus, changes in NPQ kinetics that are attributed solely to zeaxanthin should be interpreted carefully unless PsbS status and ΔpH dynamics are simultaneously measured.

Lumen acidification also enforces a rapid feedback at the main electron-transfer bottleneck: the cytochrome b6f complex. When light harvesting transiently exceeds stromal capacity to consume ATP and reducing power, ΔpH can slow plastoquinol oxidation at the Qo site (“photosynthetic control”), restricting electron transfer from PSII toward PSI and thereby reducing the probability of PSI acceptor-side over-reduction during transients (Tikkanen et al., 2015; Kono and Terashima, 2016; Degen and Johnson, 2024).

photosynthetic control reduces electron pressure (less electron delivery into PSI when sinks lag).

Both mechanisms are triggered by ΔpH, the timing and amplitude of lumen pH changes—set upstream by proton deposition, ATP synthase g(H+), and counter-ion fluxes—determine how effectively these switches engage during a sunfleck and how quickly they relax during a shadefleck (Davis et al., 2017; Degen and Johnson, 2024).

Beyond qE, additional NPQ components operate on longer timescales and should be treated as mechanistically distinct rather than as “slower qE.”

qZ (minutes): Zeaxanthin-associated quenching that overlaps with qE but persists longer as pigment state changes and relaxes more slowly than ΔpH alone (Demmig-Adams et al., 2020; Papageorgiou and Govindjee, 2014).

qI (hours): Photoinhibitory down-regulation linked to PSII damage/repair dynamics rather than to a reversible ΔpH switch; it reflects sustained stress and recovery processes (Roach and Krieger-Liszkay, 2014).