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-CF1 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. violaxanthin deepoxidase ͉ photoinhibition ͉ xanthophyll cycle ͉ proton motive force ͉ chemiosmotic coupling L ight-driven transthylakoid proton motive force (pmf ) serves two essential roles in higher plant photosynthesis (1). First, it is the central intermediate in the chemiosmotic circuit for light-driven ATP synthesis. Light-driven electron transfer leads to the pumping of protons from the stroma to the thylakoid lumen, establishing pmf, which drives the endergonic synthesis of ATP from ADP and orthophosphate (P i ) at the CF O -CF 1 ATP synthase (ATP synthase).Second, the ⌬pH component of pmf is the key regulatory signal for initiation of nonphotochemical quenching (NPQ) of excitation energy, which is important for photoprotection. Light absorption by the light-harvesting complexes (LHCs) in excess of that which can be processed can lead to harmful side reactions, collectively termed photoinhibition, that can occur at several levels, including the antenna complexes, the oxidizing and reducing sides of photosystem II (PS II), and the reducing side of photosystem I (PS I) (see reviews in refs. 2 and 3).In higher plants, photoinhibition is avoided in part by activation of NPQ, which can dump a large fraction of excitation energy, preventing the accumulation of reactive intermediates (see reviews in refs. 4 and 5-7). It is now generally accepted that NPQ involves two processes activated by acidification of the lumen, the interconversion of xanthophyll cycle carotenoids by violaxanthin deepoxidase (VDE), and the protonation of residues on key LHC components, in particular the psbS subunit (reviewed in ref. 7). Arabidopsis mutants deficient in NPQ are light sensitive, confirming its role in photoprotection (e.g., refs. 8 and 9-12).In the most basic working model for NPQ function (e.g., figure 2 of reference 12), where the kinetic and thermodynamic properties of each step in the process are consistent, NPQ should be a continuous function of linear electron flow (LEF). On the other hand, it has become clear that the relationship between LEF and NPQ is strongly modulated in response to rapid changes in physiological state, and we provide a direct demonstration of this below. It has been suggested that such NPQ mod...

In higher plant chloroplasts, transthylakoid proton motive force serves both to drive the synthesis of ATP and to regulate light capture by the photosynthetic antenna to prevent photodamage. In vivo probes of the proton circuit in wild-type and a mutant strain of Arabidopsis thaliana show that regulation of light capture is modulated primarily by altering the resistance of proton efflux from the thylakoid lumen, whereas modulation of proton influx through cyclic electron flow around photosystem I is suggested to play a role in regulating the ATP͞NADPH output ratio of the light reactions.ATP synthase proton conductivity ͉ cyclic electron flow ͉ linear electron flow ͉ energy-dependent nonphotochemical quenching ͉ protein motive force P hotosynthesis converts light energy into chemical energy, ultimately powering the vast majority of our ecosystem (1). Higher plant photosynthesis is initiated through absorption of light by antennae complexes that funnel the energy to photosystem (PS) II and I. The photosystems operate in sequence with the plastoquinone pool, the cytochrome b 6 f complex, and plastocyanin to oxidize H 2 O and reduce NADP ϩ to NADPH in what is termed linear electron flow (LEF). LEF is coupled to proton translocation, establishing a transthylakoid electrochemical gradient of protons, termed the proton motive force (pmf ) (2), comprised of electric field (⌬ ) and pH (⌬pH) gradients (3). Dual Role of the pmfThe pmf plays two central roles in higher plant photosynthesis (4). First, pmf drives the normally endergonic synthesis of ATP through the CF 1 -CF 0 ATP synthase (ATP synthase) (5). Both the ⌬pH and ⌬ components of pmf contribute to ATP synthesis in a thermodynamically, and probably kinetically, equivalent fashion (6). Second, pmf is a key signal for initiating photoprotection of the photosynthetic reaction centers through energydependent nonphotochemical quenching (q E ), a process that harmlessly dissipates excessively absorbed light energy as heat (7-10). Only the ⌬pH component of pmf, through acidification of the lumen, is effective in initiating q E by activating violaxanthin de-epoxidase, a lumen-localized enzyme that converts violaxanthin to antheraxanthin and zeaxanthin, and by protonating lumen-exposed residues of PsbS, a pigment-binding protein of the PS II antenna complex (11). A Need for Flexibility in the Light ReactionsA major open question concerns how the light reactions achieve the flexibility required to meet regulatory needs and match downstream biochemical demands (12). In LEF to NADP ϩ , the synthesis of ATP and the production of NADPH are coupled, producing a fixed ATP͞NADPH output ratio. LEF alone is probably unable to satisfy the variable ATP͞NADPH output ratios required to power the sum of the Calvin-Benson cycle (13,14) and other metabolic processes (alternate electron and ATP sinks) that are variably engaged under different physiological conditions (12,15,16). Failure to match ATP͞NADPH output with demand will lead to buildup of products and depletion of substrates for the...

Endogenous probes of light-induced transthylakoid proton motive force (pmf), membrane potential (Deltapsi) and DeltapH 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 pK(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 approximately 0.5 pH units, the lumen pH was estimated to range from approximately 7.5 (under weak light at ambient CO(2)) to approximately 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 pK(a) values for accumulation of zeaxanthin and PsbS protonation were found to be approximately 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 Deltapsi, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.

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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 6f complex were estimated by measuring the DIRK signals associated with reduction of cyt f and P700. The rates of proton pumping through the electron transfer chain and the CF O-CF1 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 b6f 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 6f complex.steady-state electron and proton transfer ͉ chemiosmotic coupling ͉ cyclic electron transfer ͉ energy budget T he energy budget of a plant depends upon the ratio of protons pumped across the thylakoid membrane to electrons passed through photosynthetic electron transfer complexes (the H ϩ ͞e Ϫ ratio), which sets the stoichiometries of ATP and NADPH production for use in the Calvin-Benson cycle and other biochemical pathways (reviewed in refs. 1 and 2). There has been a long-standing debate about the magnitude of H ϩ ͞e Ϫ for green plant photosynthesis, particularly regarding the proton pumping reactions of the cytochrome (cyt) b 6 f complex. It is generally accepted (see review in ref.3) that for each electron transferred through the linear pathway, one proton is released into the lumen at the level of water splitting; another is transported across the thylakoid membrane by the reduction and reoxidation of plastoquinone (PQ) at the photosystem II (PSII) Q B site and the cyt b 6 f complex Q o site, respectively; and a third proton is pumped, at least under some conditions, by the turnover of a Q cycle associated with the oxidation of plastoquinol at the cyt b 6 f complex (see refs. 4-7 for reviews). However, in vitro measurements of H ϩ ͞e Ϫ ratios for linear electron transport in isolated thylakoids range from 2 (e.g., refs. 8-10) to 3 (e.g., refs. 11 and 12), and several groups have suggested that it changes from 3 to 2 with increasing light intensity (1,11,(13)(14)(15)(16)(17). Such indications of variable coupling ratios have led several groups to suggest that the Q cycle or its proton pumping rea...

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (Pi), which results in down-regulation of light capture via the exciton quenching (qE) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon Pi depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CFO-CF1 ATP synthase to protons (gH + ) is decreased, retarding proton efflux from the lumen. Sequestering of Pi by mannose infiltration increased sensitivities of qE and pmf to LEF. The effects were attributable to decreases in gH + , but not to CEF1 and were largely reversed by subsequent Pi feeding. Rapid recovery of gH + in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson-Calvin cycle intermediates or lowering of stromal Pi below its KM at the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.

The formation of trans -thylakoid proton motive force ( pmf ) is coupled to light-driven electron transfer and both powers the synthesis of ATP and acts as a signal for initiating antenna regulation. This key intermediate has been difficult to study because of its ephemeral and variable qualities. This review covers recent efforts to probe pmf in vivo as well as efforts to address one of the key questions in photosynthesis: How does the photosynthetic machinery achieve sufficient flexibility to meet the energetic and regulatory needs of the plant in a varying environment? It is concluded that pmf plays a central role in these flexibility mechanisms.Key-words : CF 1 -CF 0 ATP synthase proton conductivity; cyclic electron flow around photosystem I; proton motive force.Abbreviations : CEF1, cyclic electron flow around photosystem I; cyt, cytochrome; CF 1 -CF O , chloroplast ATP synthase; D pH, pH component of pmf ; D y , electric field component of pmf ; D G ATP , the free energy of ATP formation; DIRK, dark interval relaxation kinetics; ECS, electrochromic shift; ECS t , total magnitude of ECS decay during a light-dark transition; ECS ss , steady-state ECS; ECS inv , ECS change from inverted D y ; Fd, ferredoxin; g H + , CF 1 -CF O ATP synthase proton conductivity; LEF, linear electron flow; LHCs, light harvesting complexes; n , number protons required for formation of one ATP; P 700 , primary electron donor of photosystem I; P 700 + , oxidized primary donor of photosystem I; pmf , transthylakoid proton motive force; pmf LEF , pmf generated solely by LEF; PQ, plastoquinone; PQH 2 , plastoquinol; PS, photosystem; f I , photochemical yield of photosystem I; f II , photochemical yield of photosystem II; q E , energy-dependent quenching of antenna excitons; t ECS , time constant for ECS decay in response to a brief dark interruption of steady state; v CEF1 , steady-state rate of CEF1; v H + , steady-state rate of proton flux; v LEF , steady-state rate of electron flux through LEF.

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.