Photosynthetic light reactions establish electron flow in the chloroplast's thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist-linear electron flow (LEF) from water to NADP(+) via photosystem (PS) II and PSI in series and cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b(6)f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF (Supplementary Fig. 1). Thus, formation and dissociation of the PSI-LHCI-LHCII-FNR-Cyt bf-PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

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.

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.

Plants respond to changes in light quality by regulating the absorption capacity of their photosystems. These short-term adaptations use redox-controlled, reversible phosphorylation of the lightharvesting complexes (LHCIIs) to regulate the relative absorption cross-section of the two photosystems (PSs), commonly referred to as state transitions. It is acknowledged that state transitions induce substantial reorganizations of the PSs. However, their consequences on the chloroplast structure are more controversial. Here, we investigate how state transitions affect the chloroplast structure and function using complementary approaches for the living cells of Chlamydomonas reinhardtii. Using small-angle neutron scattering, we found a strong periodicity of the thylakoids in state 1, with characteristic repeat distances of ∼200 Å, which was almost completely lost in state 2. As revealed by circular dichroism, changes in the thylakoid periodicity were paralleled by modifications in the long-range order arrangement of the photosynthetic complexes, which was reduced by ∼20% in state 2 compared with state 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna size during state 1 to state 2 transition (∼20%) is not commensurate to the decrease in PSII antenna size (∼70%), leading to the possibility that a large part of the phosphorylated LHCIIs do not bind to PSI, but instead form energetically quenched complexes, which were shown to be either associated with PSII supercomplexes or in a free form. Altogether these noninvasive in vivo approaches allow us to present a more likely scenario for state transitions that explains their molecular mechanism and physiological consequences.green algae | photosynthesis | thylakoid membrane T he efficient operation of the photosynthetic machinery of oxygenic photosynthetic organisms requires a balanced energy supply to the two photosystems (PSs) under changing environments. To avoid unbalanced excitations of the two PSs, a rapid acclimation mechanism, state transitions (STs), takes place, which allows redistributing the excitation energy between the two PSs. ST is seen in green algae and vascular plants and is modulated by the redox-controlled, reversible phosphorylation of LHCII, the light-harvesting chlorophyll a/b antenna complex. A serine-threonine protein kinase, STN7 in vascular plants (1) and Stt7 in green algae (2), is responsible for this phosphorylation. Stt7/STN7 activity depends on the redox state of the plastoquinone (PQ) pool, which is sensed by the cytochrome b 6 f complex (3).According to the current model of STs, preferential PSII excitation leads to PQ reduction, and thus to LHCII phosphorylation by the kinase. The PQ pool can also be reduced in dark anaerobic conditions in algal suspensions. In this state, called state 2 (S2), phosphorylated LHCII dissociates from PSII, thereby reducing its absorption cross-section, and associates to PSI, acting as an additional antenna for this complex. In the reverse process, prefere...