Energy-dependent quenching of excess absorbed light energy (qE) is a vital mechanism for regulating photosynthetic light harvesting in higher plants. All of the physiological characteristics of qE have been positively correlated with charge transfer between coupled chlorophyll and zeaxanthin molecules in the light-harvesting antenna of photosystem II (PSII). We found evidence for charge-transfer quenching in all three of the individual minor antenna complexes of PSII (CP29, CP26, and CP24), and we conclude that charge-transfer quenching in CP29 involves a delocalized state of an excitonically coupled chlorophyll dimer. We propose that reversible conformational changes in CP29 can “tune” the electronic coupling between the chlorophylls in this dimer, thereby modulating the energy of the chlorophyll-zeaxanthin charge-transfer state and switching on and off the charge-transfer quenching during qE.

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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...

Previous work on intact thylakoid membranes showed that transient formation of a zeaxanthin radical cation was correlated with regulation of photosynthetic light-harvesting via energy-dependent quenching. A molecular mechanism for such quenching was proposed to involve charge transfer within a chlorophyll-zeaxanthin heterodimer. Using near infrared (880 -1100 nm) transient absorption spectroscopy, we demonstrate that carotenoid (mainly zeaxanthin) radical cation generation occurs solely in isolated minor light-harvesting complexes that bind zeaxanthin, consistent with the engagement of charge transfer quenching therein. We estimated that less than 0.5% of the isolated minor complexes undergo charge transfer quenching in vitro, whereas the fraction of minor complexes estimated to be engaged in charge transfer quenching in isolated thylakoids was more than 80 times higher. We conclude that minor complexes which bind zeaxanthin are sites of charge transfer quenching in vivo and that they can assume Non-quenching and Quenching conformations, the equilibrium LHC(N) % LHC(Q) of which is modulated by the transthylakoid pH gradient, the PsbS protein, and protein-protein interactions.Higher plant photosynthesis is initiated by absorption of light in pigment-binding (antenna) proteins that transfer absorbed solar energy to the reaction centers of photosystems (PS) 3 II and I where energy conversion begins (1). The PSIIassociated light-harvesting complexes (LHCs) bind chlorophylls and carotenoids that are involved in both the harvesting and transfer of energy to the reaction center, and the harmless dissipation of excitation energy in excess of photosynthetic capacity (2). Thus, the PSII LHCs are critical branch-points for energy partitioning during photosynthesis. The peripheral antenna consists of trimeric complexes composed of LHCII proteins, the major LHC of higher plant antennae. In between the peripheral LHCII and the reaction center there are three minor LHCs referred to as CP24, CP26, and CP29 (1).Dissipation of excess light energy during photosynthesis involves several photoprotective mechanisms, which are collectively referred to as non-photochemical quenching (NPQ) (2, 3). The predominant component of NPQ is referred to as energy-dependent quenching, or qE, and it is rapidly reversible and correlated with zeaxanthin (Z) formation (4). Mutants of Arabidopsis thaliana have been instrumental in confirming the involvement of Z (5) and identifying a role for PsbS in qE (6). The npq4 mutant lacks a functional PsbS protein and exhibits very little qE (6). The PsbS protein has been subsequently proposed to be involved in controlling qE by sensing thylakoid lumen pH (7). The xanthophyll cycle consists of the enzymatic and reversible conversion of the thylakoid-associated pigment violaxanthin (V) to antheraxanthin (A) and Z (8). Very little qE is exhibited in the A. thaliana mutant referred to as npq1 which is impaired in its ability to convert V to Z as a result of a lesion in the gene encoding the thylakoid lumen-loca...

Energy-dependent exciton quenching, or qE, protects the higher plant photosynthetic apparatus from photodamage. Initiation of q E involves protonation of violaxanthin deepoxidase and PsbS, a component of the photosystem II antenna complex, as a result of lumen acidification driven by photosynthetic electron transfer. It has become clear that the response of q E to linear electron flow, termed ''q E sensitivity,'' must be modulated in response to fluctuating environmental conditions. Previously, three mechanisms have been proposed to account for q E modulation: (i) the sensitivity of q E to the lumen pH is altered; (ii) elevated cyclic electron flow around photosystem I increases proton translocation into the lumen; and (iii) lowering the conductivity of the thylakoid ATP synthase to protons (g H؉) allows formation of a larger steady-state proton motive force (pmf). Kinetic analysis of the electrochromic shift of intrinsic thylakoid pigments, a linear indicator of transthylakoid electric field component, suggests that, when CO 2 alone was lowered from 350 ppm to 50 ppm CO 2, modulation of qE sensitivity could be explained solely by changes in conductivity. Lowering both CO 2 (to 50 ppm) and O2 (to 1%) resulted in an additional increase in q E sensitivity that could not be explained by changes in conductivity or cyclic electron flow associated with photosystem I. Evidence is presented for a fourth mechanism, in which changes in q E sensitivity result from variable partitioning of proton motive force into the electric field and pH gradient components. The implications of this mechanism for the storage of proton motive force and the regulation of the light reactions are discussed. P lant chloroplasts convert light energy into two forms usable by the biochemical processes of the plant (1, 2). Redox free energy is stored by linear electron flow (LEF) through photosystem (PS) II, the cytochrome b 6 f complex, PS I, ferredoxin, and finally NADPH. Translocation of protons from the stroma to the lumen is coupled to LEF, resulting in the establishment of transthylakoid proton motive force (pmf ), which drives the synthesis of ATP from ADP and P i at the thylakoid CF o -CF 1 ATP synthase (3). It has become clear that certain redox carriers and the pmf also play regulatory roles in photosynthesis. The redox status of the electron transfer chain regulates a range of processes by means of the thioredoxin system (4) and the plastoquinone pool (5). Meanwhile, the pH component (⌬pH) of pmf regulates the efficiency of light capture by means of protonation of thylakoid lumen proteins (6). The balancing of these two roles governs the development and efficiency of the photochemical machinery, as well as the avoidance of harmful side reactions.The Need for Down-Regulation of the Photosynthetic Apparatus Plants are exposed to widely varying environmental conditions, often resulting in light energy capture that exceeds the capacity of the photosynthetic apparatus (7-10), which in turn can lead to photodamage (11,12). Plants have evolved...

Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid DpH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthinless1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and a-carotene than the wild type. szl1 contains a point mutation in the lycopene b-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene b-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.

Estimation of the maximum chlorophyll fluorescence yield under illumination, or FmЈ, by traditional single saturation pulse (SP) methodology is prone to underestimation error because of rapid turnover within photosystem (PS) II. However, measurements of fluorescence yield during several single pulses of variable intensity describes the irradiance dependence of apparent FmЈ, from which estimates of FmЈ at infinite irradiance can be derived. While such estimates have been shown to result in valid approximations of FmЈ, the need to apply several single pulses limits its applicability. We introduce a novel approach that determines the relationship between apparent FmЈ and variable irradiance within a singlẽ 1 s multiphase flash (MPF). Through experiments and simulations, we demonstrate that the rate of variation in irradiance during an MPF is critical for achieving quasisteady-state changes in the proportions of PSII acceptor side redox intermediates and the corresponding fluorescence yields, which are prerequisites for accurately estimating FmЈ at infinite irradiance. The MPF methodology is discussed in the context of improving the accuracy of various parameters derived from chlorophyll fluorescence measurements, such as photochemical and non-photochemical quenchings and efficiencies. The importance of using MPF methodology for interpreting chlorophyll fluorescence, in particular for integrating fluorescence and gas exchange measurements, is emphasized.

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.