Determining the limitations and regulation of photosynthetic energy transduction in leaves

Transcription

1 Plant, Cell and Environment (2007) 30, doi: /j x Determining the limitations and regulation of photosynthetic energy transduction in leaves NEIL R. BAKER 1, JEREMY HARBINSON 2 & DAVID M. KRAMER 3 1 Department of Biological Sciences, University of Essex, Colchester, CO4 3SQ, Essex, UK, 2 Horticultural Production Chains Group, Wageningen University, Marijkeweg 22, Wageningen 6709 PG, the Netherlands and 3 Institute of Biological Chemistry, 289 Clark Hall, Washington State University, Pullman, WA , USA ABSTRACT The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo. Key-words: ATP synthesis; chlorophyll fluorescence; cyclic electron flux; electrochromic shift; electron transport; lightinduced absorbance change; linear electron flux; photosystem I; photosystem II; proton transport. Abbreviations: CEF1, cyclic electron flux around PSI; DIRK, dark interval relaxation kinetics; ETR, electron transport rate; LEF, linear electron flux; MDA, monodehydroascorbate; NADP MDH, NADP malate dehydrogenase; P680, reaction centre chlorophyll of PSII; P700, reaction centre chlorophyll of PSI; P700 +, oxidized P700; P700 0, non-oxidized P700; PAR, photosynthetically active radiation; pmf, transthylakoid proton motive force; PPFD, photosynthetically active photon flux density; PQ, plastoquinone; PQH 2, plastoquinol (reduced plastoquinone); Q A, bound primary plastoquinone electron acceptor of PSII; Q B, bound secondary plastoquinone acceptor of PSII; DG ATP, free energy of ATP formation; Correspondence: N. R. Baker. Fax: ; baken@essex.ac.uk DpH, ph component of pmf; DY, electric field component of pmf; t ECS, time constant for ECS decay after a brief dark interruption of light steady state; F PSI, quantum efficiency of PSI electron transport. INTRODUCTION The major role of the photosynthetic apparatus of higher plant thylakoids is to transduce light energy into ATP and reductants (usually NADPH). Light is captured by an array of light-harvesting complexes, which absorb light and transfer excitation energy to the reaction centres of photosystem I (PSI) and photosystem II (PSII) to drive the primary photochemical reactions and create a separation of electrical charge. These light-driven charge separations at PSI and PSII effectively drive electron flux from water to terminal electron acceptors. The linear electron flux (LEF) from water through the PSII and PSI reaction centres is coupled to H + release during water oxidation and the shuttling of H + across the thylakoid membrane, which together establish a H + electrochemical potential difference, or pmf, across the thylakoid membrane. A cyclic electron flux around PSI (CEF1) can also result in H + transfer from the stroma to lumen, and contribute to the pmf. The pmf can be used to drive ATP synthesis by the transport of H + through the ATP synthase back into the stroma. A schematic drawing outlining these processes is shown in Fig. 1. The composition, structure and functions of the photosynthetic apparatus of the thylakoid have been extensively studied and are now very well understood. In mature leaves, the primary role of the thylakoid photosynthetic apparatus is to provide ATP and reductants to meet the metabolic requirements for carbon assimilation and other energyrequiring processes. In the natural environment, rates of carbon assimilation by leaves can fluctuate markedly because of fluctuations in irradiance, or as the leaves experience various stresses, such as drought and high temperature, which restrict carbon assimilation. In situations where irradiance is limiting, efficiency needs to be maximized, placing a premium on efficient energy transfer to the reaction centres. When irradiance is not limiting, increases in LEF could produce increases in ATP and NADPH production that could thermodynamically or kinetically constrain LEF and result in over-excitation of the reaction centres, which could potentially lead to photodamage. Journal compilation 2007 Blackwell Publishing Ltd 1107

2 1108 N. R. Baker et al. Figure 1. Schematic representation of the electron (orange arrows) and proton transfers (blue arrows), and associated processes that can occur as a result of light absorption by the thylakoid photosystems. Excitation of photosystem II (PSII) and photosystem I (PSI) oxidizes their reaction centres and drives LEF from water to NADPH (upper side of thylakoid). Electron flux from PSII and proton uptake from the stroma reduce PQ to PQH 2. From PQH 2, half of the electrons are transferred via the cytochrome b 6f complex and plastocyanin (Pc) to PSI, which then transfers the electrons via ferredoxin (Fd), and a ferredoxin NADP oxidoreductase to NADP resulting in the generation of NADPH. The other half of the electrons from PQH 2 is returned via the cytochrome b 6f complex to PQ. Excitation of PSI can result in CEF around PSI via ferredoxin, PQ, cytochrome b 6f complex and Pc (lower side of thylakoid). Reduction of PQ by ferredoxin is mediated by a plastoquinone reductase (PQR). Oxidation of water by PSII and PQH 2 by the cytochrome b 6f complex releases protons into the lumen creating a pmf across the membrane. Proton buffering will initially result in storage of the pmf as electric field (Dy). However, the Dy will be collapsed by counterion movements; with continued H + influx into the lumen, the buffering capacity will be exceeded and the ph component (DpH) of the pmf will be formed. Movement of H + from the lumen to the stroma via the ATP synthase results in ATP formation. The rate of electron flux from PQH 2 through the cytochrome b 6f complex I negatively regulated by an increasing pmf. The excitation density within PSII antennae can be regulated by energy-dependent quenching (q E; shown as loss of heat by a brown arrow). Creation of q E is associated with the ph-dependent activation of violaxanthin de-epoxidase (VDE), which reduces violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z), and the protonation of the Psbs protein. Consequently, excitation dissipation via q E is dependent on the accumulation of H + in the lumen. Consequently, it is essential that the delivery of excitation energy to the reaction centres is regulated to prevent such damage, and this is achieved by the photosynthetic energy transduction systems having extremely flexible constraints on their efficiency. Besides using ATP and reductants for carbon assimilation, leaves use these photosynthetic products for a range of other metabolic processes, for example, nitrogen and sulphur metabolism, biosynthesis of macromolecules, which can differ in their stoichiometric requirements for ATP and reductants. Under suboptimal environmental conditions that reduce the ability to assimilate carbon, the proportion of electrons being consumed by such other electron sinks can increase markedly. Thus, besides having to regulate the rate of excitation of the reaction centres when CO 2 assimilation is restricted, leaves also have to regulate the ratio of ATP to NADPH being produced by the thylakoid photosynthetic apparatus. The regulation of the rate of excitation of the reaction centres and the ratio of ATP/NADPH production involves many factors associated with LEF and H + transport in the thylakoid. In this review, we examine how information

3 Photosynthetic light energy transduction in leaves 1109 about PSI, PSII and H + transport can be obtained and used to provide insights into the regulation of light energy transduction by thylakoids in vivo. Techniques that provide such information are described, and these allow analysis of the factors involved in regulation of light use and electron transport. Each of the techniques yields its own set of parameters. In a simple application, it may be that only one technique need be employed, for example, measurement of photodamage to PSII can be done using chlorophyll fluorescence alone. However, in many cases, particularly when examining the response of photosynthesis to the environment, a thorough analysis of limitations will require the concurrent application of several techniques. Correlation of the results from these different techniques allows identification of limiting processes and the regulation of fluxes. The review concludes with a summary of some of the current challenges in understanding the complexities of regulation. PSII ELECTRON FLUX Excitation of PSII results in oxidation of water and reduction of PQ. The excited reaction centre, P680*, can rapidly transfer an electron to a primary acceptor, pheophytin (Pheo), which will then transfer the electron to a bound plastoquinone (Q A). Q A- will then reduce a second plastoquinone (Q B). P680 + is re-reduced by electron transfer from a tyrosine residue (Y z), which is re-reduced as a result of water oxidation. A second electron transfer from P680* to Q B-, coupled with its protonation from the stroma, results in the formation of PQH 2, which then dissociates from the PSII complex. Consequently, PSII acts as a water plastoquinone oxidoreductase. PQH 2 can now transfer the two electrons to the cytochrome b 6f complex, and release the two protons into the thylakoid lumen, thus contributing to the formation of a H + electrochemical potential difference across the thylakoid membrane. Measurement of PSII electron transport Modulated chlorophyll fluorescence measurements are widely used to estimate the quantum efficiency of PSII photochemistry and provide insights into the regulation of the rate of excitation of PSII reaction centres in leaves (Baker & Oxborough 2004). A leaf in continuous actinic light when monitored with a weak modulated fluorescence excitation beam has a fluorescence yield of F, which rises to a maximal level F m when the leaf is exposed to a saturating pulse of actinic light that maximally reduces Q A. The difference between F m and F, designated F q, is the result of quenching of fluorescence by PSII photochemistry (Baker & Oxborough 2004). Genty, Briantais & Baker (1989) showed that F q /F m was theoretically proportional to the quantum yield of PSII photochemistry, and this was confirmed empirically from direct measurements of oxygen evolution using mass spectrometry (Genty et al. 1992). Clearly, this fluorescence parameter could be used to provide a rapid and effective way of estimating the quantum efficiency of LEF through PSII in leaves under different conditions (hereafter referred to as the PSII operating efficiency); this parameter has previously been termed DF/F m and F PSII in the literature. Assuming that a constant proportion of the reductants resulting from LEF is utilized for CO 2 assimilation, then the PSII operating efficiency would be predicted to be directly proportional to the operating quantum efficiency of CO 2 assimilation (Genty et al. 1989; Baker & Oxborough 2004). Empirically, this has been shown in leaves where photorespiration is absent or suppressed (Genty et al. 1989; Genty, Harbinson & Baker 1990a; Harbinson, Genty & Baker 1990; Krall & Edwards 1990, 1991; Cornic & Ghashghaie 1991; Krall et al. 1991; Edwards & Baker 1993; Siebke et al. 1997), and demonstrates the robustness of the use of fluorescence to investigate LEF through PSII. A list of the major fluorescence parameters used in analysis of PSII photochemistry is given in Table 1, together with their definitions and a brief explanation of their physiological relevance. Because the PSII operating efficiency is directly related to the rate of LEF, it is possible in theory to estimate the rate of non-cyclic electron transport through PSII (ETR) using the following equation: ETR = I( A )( fraction )( F F ), (1) PSII leaf PSII q m where I is the incident PPFD on the leaf, A leaf is the spectral absorbance of the leaf and fraction PSII is the fraction of incident photons that are absorbed by PSII. It is frequently assumed that leaves absorb 84% of incident photons, and that 50% of these photons are absorbed by PSII, and consequently Eqn 1 is often modified to: ETR = 042. I( F F ), (2) q m This equation is routinely used by commercial modulated fluorometers to estimate ETR. Although the assumption that leaves absorb 84% of incident photons is reasonably accurate for most mature green leaves, it is not always the case, and large deviations from this value can occur (Hodáňová 1985; Ehleringer 1991; Jones 1992). Similarly, the assumption that 50% of the light absorbed by the leaf is absorbed by PSII may be reasonable in many cases, but there will be many situations where this is not the case. The absorptivity of a leaf can be accurately determined using an integrating sphere with an appropriate light source and a spectroradiometer, although the effect on efficiency of nonphotosynthetic blue-light absorbing pigments present in many leaves cannot be corrected for by means of a simple absorption measurement. However, it is very difficult to determine accurately the fraction of absorbed photons that are reaching PSII. Consequently, caution must be exercised when attempting to estimate ETR from measurements of PSII operating efficiency. Another important potential source of error when using F q /F m to estimate ETR is the contribution of PSI fluorescence emission to measured

4 1110 N. R. Baker et al. Table 1. Definitions of the major modulated chlorophyll fluorescence parameters used in studies of PSII photochemical performance Fluorescence parameter Definition Physiological relevance F, F Fluorescence emission from dark- or light-adapted leaf, respectively. Provides little information on photosynthetic performance as they are influenced by many factors F o, F o Minimal fluorescence from dark- and light-adapted leaf, respectively Level of fluorescence when PSII primary quinone electron acceptors are maximally oxidized (PSII centres open ) F m, F m Maximal fluorescence from dark- and light-adapted leaf, respectively Level of fluorescence when Q A is maximally reduced (PSII centres closed ) F v, F v Variable fluorescence from dark- and light-adapted leaves, respectively Demonstrates ability of PSII to perform primary photochemistry F q Difference in fluorescence between F m and F Photochemical quenching of fluorescence caused by open PSII centres F v/f m Maximum quantum efficiency of PSII photochemistry Maximum efficiency at which light absorbed by PSII is converted to chemical energy (Q A reduction) F q /F m PSII operating efficiency Estimates the efficiency at which light absorbed by PSII is used for photochemistry (Q A reduction); at a given light intensity, it provides an estimate of the quantum efficiency of linear electron transport through PSII; has previously been termed DF/F m and F PSII in the literature F v /F m PSII maximum efficiency Provides an estimate of the maximum efficiency of PSII photochemistry at a given light intensity, which is the PSII operating efficiency if all the PSII centres were open (Q A oxidized) F q /F v PSII efficiency factor Non-linearly related to the proportion of PSII centres that are in the open state (with Q A oxidized); relates the PSII maximum efficiency to the PSII operating efficiency; mathematically identical to the coefficient of photochemical quenching, q p q L Fraction of PSII centres which are in the open state Parameter estimating the fraction of PSII centres in open state (with Q A oxidized) based on a lake model for the PSII photosynthetic apparatus; equates to (F q /F v )(F o /F ) NPQ Non-photochemical quenching Estimates the non-photochemical quenching from F m to F m ; monitors the apparent rate constant for non-radiative decay (heat loss) from PSII and its antennae q E Energy-dependent quenching Associated with a light-induced development of DpH across the thylakoid membrane. Regulates the rate of excitation of PSII reaction centres q I Photoinhibitory quenching Associated with a photoinhibition of PSII photochemistry q T Quenching associated with a state transition Associated with phosphorylation of LHCII fluorescence parameters (Baker & Oxborough 2004). As PPFD is increased, and F q /F m decreases the relative contribution of PSI, fluorescence increases and will result in decreases in F q /F m that are not associated with changes in PSII photochemistry and consequently ETR will be underestimated. Such errors can be minimized by measuring fluorescence associated with the 683 nm emission peak of PSII where the relative contribution of PSI fluorescence is minimized (Genty et al. 1990b; Pfündel 1998; Lawson et al. 2002; Itoh & Sugiura 2004). The use of short measuring wavelengths, however, is associated with greater reabsorption of the fluorescence with the result that the measurement is now biased towards the upper surface of the leaf (Lawson et al. 2002). With longer measuring wavelengths, there is still a bias in measurements, but this is not caused by reabsorption but to the greater penetration of the excitation light into the leaf. Commercial fluorometers often measure fluorescence primarily above 700 nm, and consequently have a high probability of PSI fluorescence and cells deeper within the leaf making a significant contribution to the fluorescence detected than when shorter measuring wavelengths are used. Factors determining PSII operating efficiency The PSII operating efficiency is the product of two important fluorescence parameters, F v /F m and F q /F v, where F v is the variable fluorescence yield of a light-adapted leaf (Genty et al. 1989). F v /F m estimates the maximum quantum efficiency of PSII photochemistry in the illuminated leaf when Q A is maximally oxidized and can be used to assess the contributions of non-photochemical quenching (NPQ)

5 Photosynthetic light energy transduction in leaves 1111 F q '/ F m ', F v '/ F m ', F q '/ F v ' F v '/ F m ' F q '/ F v ' Time (min) F q '/ F m ' Figure 2. Changes in the photosystem II (PSII) operating efficiency (F q /F m, ), maximum PSII quantum efficiency (F v /F m, ) and the fraction of the maximum PSII efficiency that is realized in the light (F q /F v, ) during induction of photosynthesis in a dark-adapted mature maize leaf on exposure to a PPFD of 815 mmol m -2 s -1 over 60 min. Drawn from data of Oxborough & Baker (1997). to changes in the PSII operating efficiency (Baker & Oxborough 2004). F q /F v estimates the fraction of the maximum PSII operating efficiency that is realized in the leaf under the environmental conditions during the measurement; this relates to the proportion of PSII reaction centres with Q A oxidized, i.e. the fraction of PSII centres that are open, and is mathematically identical to the frequently used coefficient of photochemical quenching, q p. Note, however, that because of interconnectivity between PSII photosynthetic units, the quantitative relationship between Q A redox state and F q /F v (and q p) is non-linear, so changes in F q /F v can only be used to imply qualitative changes in Q A redox state. By measuring both F v /F m and F q /F v in situations where changes are occurring in the PSII operating efficiency, it is possible to evaluate whether changes in LEF through PSII are attributable to changes in NPQ or the ability of an excited PSII reaction centre to perform photochemistry. A good example of this is during the induction of photosynthesis when a dark-adapted maize leaf is exposed to light (Fig. 2). Large changes in the PSII operating efficiency are observed, which are clearly primarily associated with changes in F q /F v as only small changes in F v /F m are observed during the induction. Clearly, the fluctuations in PSII electron transport during induction of photosynthesis are almost entirely associated with changes in the ability to utilize the products of electron transport and not with changes in NPQ modifying the rate of excitation of PSII reaction centres. When light intensity is increased, the steady-state PSII operating efficiency decreases and is accompanied by decreases in both F q /F v and F v /F m (Fig. 3). The increases in NPQ, indicated by F v /F m, are saturated at much lower light intensity than the decreases in F q /F v, indicating that changes in NPQ are not contributing significantly to the large decreases in the PSII operating efficiency at higher light levels. Consequently, decreases in the PSII operating efficiency once NPQ is saturated are being determined by the ability of PSII reaction centres to transfer electrons to secondary electron acceptors. Confusingly, NPQ, in addition to being an abbreviation for a physiological process, is also used as a physiological parameter (we will use the italicized abbreviation NPQ to indicate the physiological parameter), which equates to (F m/f m ) - 1 (Bilger & Björkman 1990). This term is frequently used to assess levels of NPQ, and changes in NPQ are non-linearly related to and rise to higher values than F v /F m for a given change in NPQ (Fig. 3). However, NPQ does not allow direct evaluation of the proportion of the change in the PSII operating efficiency that is attributable to changes in NPQ, whereas F v /F m does. Furthermore, it should be noted that NPQ compares the NPQ from dark-adapted leaf at F m to F m in the light-adapted leaf. Consequently, care must be taken to only compare NPQ values from samples that have similar quenching characteristics in the dark-adapted state. A more detailed analysis of changes in the light-induced, down-regulatory quenching processes and other basal, nonphotochemical losses in PSII that occur in dark-adapted leaves caused by non-radiative decays can be made (Hendrickson, Furbank & Chow 2004; Kramer et al. 2004d). This involves calculation of the quantum yields of NPQ (F NPQ) and basal non-radiative decays (F NO), and allows accurate estimation of the fraction of excitons that are lost in down-regulatory quenching and in basal, non-radiative decay processes. NPQ can comprise of three forms of quenching: energy-dependent quenching (q E), photoinhibitory quenching (q I) and state transition-related quenching (q T), with q E and q I being the major contributors in higher plants. The majority of q E is considered to be associated with quenching in the PSII antennae resulting from the development of a large DpH across the thylakoid membrane, which activates the violaxanthin de-epoxidase that converts violaxanthin to zeaxanthin (Demmig-Adams & Adams 1996; Yamamoto, Bugos & Hieber 1999), and also results in the protonation of carboxylic acid residues of the F q '/ F m ', F v '/ F m ', F q '/ F v ', q L q L F q '/ F m ' PPFD (mmol m 2 s 1 ) NPQ F v '/ F m ' F q '/ F v ' NPQ Figure 3. Changes in the photosystem II (PSII) operating efficiency (F q /F m, ), maximum PSII quantum efficiency (F v /F m, ), the fraction of the maximum PSII efficiency that is realized in the light (F q /F v, ), the fraction of PSII reaction centres that are open (q L, ) and non-photochemical quenching (NPQ, ) as a function of PPFD in a tobacco leaf that was maintained in an atmosphere containing 100 mmol mol -1 CO 2 and 2% O 2 to reduce CO 2 assimilation and eliminate photorespiration, respectively. Drawn from data of Kramer et al. (2004d).

6 1112 N. R. Baker et al. PsbS, a protein associated with the PSII antenna (Li et al. 2000, 2004). Protonation of PsbS and binding of zeaxanthin result in conformational changes in the PSII antennae that are associated with increases in the quantum yield of thermal dissipation of excitation energy (Krause & Jahns 2004; Pascal et al. 2005). PsbS protonation and violaxanthin de-epoxidase activation depend on the intrathylakoid (also known as the lumenal) ph, so factors that influence the steady-state value of this parameter are important in determining the extent of q E (see section on Proton Fluxes). Thermal dissipation plays an important regulatory role in regulating the rate of excitation of PSII reaction centres. Attempts have been to resolve q E from q I and q T on the basis of differences in their relaxation kinetics in the dark (Horton & Hague 1988; Quick & Stitt 1989; Walters & Horton 1991); however, caution should be exercised when attempting to do this as the rate of relaxation of these quenching components can be changed by the saturating light pulses under certain conditions and differ in response to long-term environmental stresses. The relationship of F q /F v with the redox state of Q A is complex and depends on whether excitation energy transfer can occur between PSII reaction centres and the amount of NPQ at PSII (Baker & Oxborough 2004; Kramer et al. 2004d). F q /F v is linearly related to the redox state of Q A only if each PSII reaction centre has its own independent antenna system which cannot transfer excitation to the antennae of other reaction centres (Baker et al. 2001; Kramer et al. 2004d). It is clear that in leaves, this is not the case; excitation energy in PSII antennae can be competed for by a number of reaction centres (Lavergne & Trissl 1995), and F q /F v is not linearly related to the fraction of PSII centres that are open (Baker & Oxborough 2004; Kramer et al. 2004d). Assuming a lake model for the photosynthetic apparatus where PSII photosynthetic units are connected and excitation energy can be competed for by a number of reaction centres, Kramer et al. (2004d) have demonstrated that the parameter q L, which is given by (F q /F v )(F o /F ), is linearly related to the redox state of Q A. Consequently, q L can be used to monitor changes in the fraction of PSII centres that are open. For leaves exposed to a wide range of PPFDs, q L was consistently found to be lower than F q /F v, and at high PPFDs, F q /F v was almost two times greater than q L (Kramer et al. 2004d), although the pattern of change in both parameters as a function of increasing PPFD was similar (Fig. 3). PSI ELECTRON FLUX PSI is associated with the formation of the reductants required for much of the metabolism that occurs in the stroma. PSI photochemistry is initiated by excitation energy transfer from antennae pigments to the reaction centre chlorophyll, P700. The intrinsically unstable, excited state of this pigment, P700*, is a powerful reductant that can reduce the primary acceptor of PSI, A 0 (a pair of chlorophyll molecules symmetrically arranged within the PSI heterodimer) to form the strongest, stable, biologically generated reductant so far identified. The other product of this reaction is P700 +, an oxidant. From A 0, the electron is transferred to one of a pair of A 1 (phylloquinone), F x, F A and F B (iron sulphur centres) before it leaves the PSI complex to reduce ferredoxin. Ferredoxin is a mobile, water-soluble protein containing an iron sulphur centre that distributes electrons received from PSI to a diverse range of electron acceptors in the chloroplast stroma, of which quantitatively the most important is normally NADP. Meanwhile, P700 + is reduced by electron transfer from plastocyanin, which in turn receives electrons from the cytochrome b 6f complex. There are two sources for the electrons that reduce the cytochrome b 6f complex. They may be derived from PSII giving rise to the linear electron transport pathway, or they may be transferred from ferredoxin giving rise to a cyclic electron pathway. The relative contribution of these pathways is variable depending on the type of photosynthetic organism and the regulatory state of the electron transport chain. In C 3, photosynthetic organisms whose main photosynthetic activity is CO 2 assimilation and/or photorespiratory O 2 reduction, PSII is the predominant source of electrons that ultimately reduce P700. A high yield of PSIdriven CEF1 in such plants would be inconsistent with their high yield of CO 2 fixation under non-photorespiratory conditions (Genty & Harbinson 1996). Thus, in this type of system, linear electron transport is dominant and the electron transport activities of PSII and PSI are tightly coupled. In the bundle sheath cells of C 4 plants, organisms with more flexible metabolism (e.g. green algae), and possibly, stressed C 3 leaves, CEF1 can be a significant, or even the dominant, form of photosynthetic energy capture (Harbinson & Foyer 1991; Finazzi et al. 2002; Romanowska et al. 2006). Ultimately, any conditions that prevent the flow of electrons away from P700* or into P700 + will block the PSI electron flux. To sustain electron flux through PSI, the following requirements must be met: (1) there must be molecules of P700 which can be photochemically oxidized; (2) an electron transport chain that is capable of transferring the electron from P700 to ferredoxin; (3) an electron donor system receiving electrons via either the linear or cyclic pathways that can re-reduce P700 + ; and (4) metabolism (or a non-metabolic electron acceptor activity, such as O 2 reduction) that will reoxidize reduced ferredoxin. A limitation of any of these requirements will decrease the light-use efficiency of PSI. Measurement of PSI electron transport Measurements of PSI electron transport are often focused on analyzing to what extent, and by which means, donor and acceptor side processes limit PSI electron transport, and the relative contributions of linear and cyclic fluxes to the regeneration of P700 from P Unlike PSII fluorescence, the yield of fluorescence from PSI is largely considered to be unaffected by the state of the PSI reaction centre in vivo at room temperature, so fluorescence cannot be used to measure PSI electron transport in vivo (Lavorel & Etienne 1977; Itoh & Sugiura 2004). Instead, the operation of PSI in

7 Photosynthetic light energy transduction in leaves 1113 vivo is monitored by means of a light-induced absorbance change, usually in the range nm (Harbinson & Woodward 1987; Schreiber, Klughammer & Neubauer 1988). In this spectral region, the oxidation of P700 to P700 + creates an increase in absorbance. Scattering of the measuring beam by the leaf tissue increases its path length (Rühle & Wild 1979), so the absorbance increase is greater than that expected from the extinction coefficient of the absorbance change and the concentration of P700 present in leaves. This makes it impossible to use an unadjusted absorbance change to quantify the total amount of P700 in the leaf or otherwise use the absorbance change as an absolute measure of P700 oxidation. To circumvent this limitation, the absorbance increase developed during irradiance is calibrated by comparing it to the absorbance change produced during a far-red irradiance (around 720 nm) which will oxidize most of the (typically around 90%) P700 in the leaf. The far-red irradiance may also be combined with a flash of broad-band irradiance to ensure complete oxidation of the P700 pool (Kingston-Smith, Harbinson & Foyer 1999). The quantum yield of a PSI complex is zero when its P700 is oxidized; under these conditions, the reaction centre quenches the excitation energy, converting it to heat. So, in the case where there is no limitation of P700 oxidation caused by a shortage of electron acceptors, the relative amount of the P700 pool that is non-oxidized (P700 0 )isa measure of the F PSI, and is calculated from: 0 0 Φ PSI = P700 ( P700 + P700 + ) It is important to note that this is strictly a relative quantum efficiency; it is not known with certainty what the quantum yield of PSI electron transport is in absolute terms when no P700 is oxidized, although it is generally expected to be in the order of 0.95 (Lavergne & Trissl 1995). Thus the error implied by taking the relative yield to be an absolute yield is in most cases not significant. If P700 oxidation in some PSI reaction centres is limited by a shortage of electron acceptors, the measurement and calculation of PSI electron transport are more complicated because it is necessary to account for the effects of donor and acceptor side limitation (Klughammer & Schreiber 1994; Holtgrefe et al. 2003). For a wild-type (WT) leaf photosynthesizing in air, with open or closed stomata, or in 2% O 2 with CO 2 concentrations above 100 ppm, it is very unlikely that any acceptor limitation will be present, except transiently (e.g. following a large increase in irradiance). Most steady-state measurements of PSI electron transport do not, therefore, need to account for acceptor side limitation, and data obtained under these conditions are simpler to interpret in terms of changes in the quantum yield for PSI electron transport. In leaves in darkness and at irradiances where photosynthesis is completely light limited, the relative amount of P700 that is in the non-oxidized state is 100%, and the efficiency of PSI electron transport is calculated to be 1 (Fig. 4). This implies that under completely light-limited conditions, electron transport into PSI is sufficient to reduce all photochemically oxidized P700. Over most of the PAR (3) Figure 4. A typical relationship between the quantum efficiency for electron transport by PSI (F PSI) and irradiance. The data were obtained from the leaf of a tropical epiphyte Juanulloa aurantiaca photosynthesizing in air and subjected to a regime of increasing irradiance. F PSI was calculated using Eqn 3, so the efficiency is relative and uncorrected for the actual maximum efficiency of PSI, although this is expected to be 0.95 or higher (see text). spectrum, the excitation of PSII has been calculated to exceed that of PSI (Evans 1987); and in most leaves, the operating efficiency of PSII decreases sharply from the dark-adapted value of about 0.8 by about at PPFDs below 100 mmol m -2 s -1 because of reduction of Q A (see Fig. 3) (Genty et al. 1989), consistent with PSII electron transport being limited by a lack of electron acceptors (P700 + ).This decrease of PSII efficiency under light-limiting conditions implies a loss of overall light-use efficiency. There are situations where F PSI will decrease sharply at low irradiances; for example, acute photodamage to PSII can reduce the activity of PSII to a point where electron transport from PSII is insufficient to reduce photochemically generated P700 + (Genty et al. 1990a), and some mutations that diminish the amount of chlorophyll b also produce the same effect by reducing the rate of excitation of PSII reaction centres (unpublished observations). Irradiance with wavelengths that preferentially excite PSI (far red: >700 nm), or following treatment with herbicides that affect PSII electron transport, will likewise produce an increase in the steady-state pool of P700 + at low irradiances and thus decrease F PSI (Harbinson & Woodward 1987). This implies that a comparison of PSI and PSII efficiencies under strictly light-limited irradiances can provide information about the balance of excitation of the two photosystems or of damage to the photosystems. In the absence of an acceptor side limitation, increasing irradiance results in a sigmoidal decrease in the quantum yield of PSI (Fig. 4). The sensitivity of F PSI to light intensity varies between leaves and also depends on the environmental and physiological conditions of the leaf at the time of

8 1114 N. R. Baker et al. measurement, for example, temperature, drought stress, leaf age, source/sink balance, CO 2 and O 2 concentration (Harbinson, Genty & Foyer 1990; Peterson 1991; Harbinson 1994; Laisk & Oja 1994). Decreases in F PSI at constant irradiance will also be produced by factors that decrease photosynthesis, such as decreasing CO 2 concentration, decreasing temperature and drought. Although electron transport may be limited by metabolic processes, under steady-state conditions this limitation does not usually act directly to limit PSI electron transport on its acceptor side. In response to limited metabolic activity, electron transport is considered to be limited largely at the cytochrome b 6f complex (Laisk & Oja 1994; Genty & Harbinson 1996). It is, however, important to remember that the extent to which a decrease in the potential rate of electron transport through the cytochrome b 6f complex will limit electron transport as a whole will depend on irradiance. At low irradiance, where photosynthesis is limited by light-capture, inhibition of the cytochrome b 6f complex has very little effect on the rate of electron transport, whereas at saturating irradiance electron transport is much more sensitive to inhibition of the cytochrome b 6f complex (Heber, Neimanis & Dietz 1988). In contrast, electron transport at low irradiance is more sensitive to inhibition at Q B than it is at high irradiance (Heber et al. 1988). NPQ of PSII will diminish the rate of reduction of the Q A pool, and will increase with increasing irradiance above the region of light limitation of photosynthesis. By analogy with the effect of inhibition at Q B on electron transport, it is possible that NPQ could exert a weak limitation on electron transport in the range of irradiances between complete light limitation and complete light saturation, but this remains to be demonstrated. In the absence of acceptor side limitations, F PSI can be used to estimate the electron flux through PSI (ETR PSI, also often termed J PSI): ETR = I( A )( fraction )( Φ ) (4) PSI leaf PSI PSI where I is the incident PPFD on the leaf, A leaf is the spectral leaf absorptance and fraction PSI is the fraction of the absorbed irradiance that is trapped by PSI complexes. It is difficult to determine fraction PSI experimentally, and consequently it is often assumed to be 0.5. As is the case for PSII (see previous text), the assumption that 50% of the photons absorbed by the leaf are absorbed by PSI will frequently be incorrect. Kinetics of P700 + reduction Removal of irradiance from a leaf results in the reduction of P700 +, and analysis of the kinetics of this reduction can be used to provide information on the regulation of F PSI. In the absence of regulation on the donor side, an increase in PSI electron transport, for example, produced by increasing irradiance, would be limited by the approach to redox equilibrium between electron donors and acceptors on the acceptor side of PSI. This would result in the increasing reduction of the electron acceptor pool of the stroma to the Absorbance change (at 820 nm, arbitrary units) Stedady-state level of photooxidized P700 (P700 + ) Irradiance off P reduction in the dark Complete P reduciton Time (s) Figure 5. Typical decay kinetics of the absorption change at 820 nm (DA 820) from a leaf produced by removing the irradiance. In the absence of irradiance, the steady-state of P700 oxidation and P700 + reduction is unbalanced by the absence of oxidation, and the pool of P700 + decays to zero following kinetics determined by the rate constant for electron transport from PQH 2 and the cytochrome b 6f complex. In addition to the millisecond time-scale kinetics shown in this figure, more rapid (sub-millisecond) kinetics of P700 + reduction will occur because of electron transfer from the fraction of pools of plastocyanin and cytochrome f that were already reduced at the point of cessation of irradiance. These kinetics, which increasingly dominate as the irradiance is decreased, are unresolved in this measurement. point that forward electron transport from P700 would become impossible. Electron transport processes in the reaction centre would then be dominated by back reactions (Rutherford & Heathcote 1985). However, in vivo, the stroma does not become extensively reduced except transiently or under extreme conditions, for example, at CO 2 concentrations below 100 ppm when the O 2 concentration is 2% (Takahama, Shimuzu-Takahama & Heber 1981; Dietz & Heber 1984; Harbinson et al. 1990; Foyer, Lelandais & Harbinson 1992). The primary limitation of PSI electron transport therefore largely resides on the donor side at the cytochrome b 6f complex rather than the acceptor side, even when metabolic demand for reductant is low. The rate of electron transport from the PQH 2 pool to the cytochrome b 6f complex is subject to short- (Tikhonov, Khomutov & Ruuge 1984; Nishio & Whitmarsh 1993) and long- (Onoda, Hikosaka & Hirose 2005) term control; short-term control is effected by changes in intrathylakoid ph, whereas longterm control is caused by changes in the amount of the cytochrome b 6f complex. It is relatively easy to measure the extent of the controlled donor side limitation of electron transport by measuring the reduction kinetics of P700 + after the irradiance is removed. When irradiance is removed from the leaf, the rate of P700 oxidation falls to zero and the rate constant for P700 + reduction can then be obtained from the pseudo-first-order decay of the DA 820 absorbance change (Fig. 5). This decay, which has a half-time of 3 4 ms

9 Photosynthetic light energy transduction in leaves 1115 or greater, reflects the rate-limiting supply of reductant passing from PQH 2 via the cytochrome b 6f complex and plastocyanin to P The rate constant for this supply of reductant to P700 +, k e, is a measure of the capacity for electron transport via this rate-limiting mechanism and can be treated like a conductance in leaf gas exchange models. A valuable feature of k e is that it is absolute, not relative like F PSI. This allows comparisons to be made between leaves and for the basis of changes in the relationship between F PSI and PPFD relationship to be analysed in terms of changes in k e (Riethmuller-Haage et al. 2006). Attempts to measure changes in the rate of electron transport into P700 + by measuring the initial slope of the millisecond decay component (Johnson 2005) were in error because they ignored the sub-millisecond kinetics caused by transfer from the reduced plastocyanin and cytochrome b 6f pools which are not resolvable with instruments with a measuring beam-modulation frequency of 100 khz (Sacksteder & Kramer 2000; Kramer et al. 2004a). The error will be greatest at lower PPFDs where the size of the reduced plastocyanin and cytochrome b 6f pools will be greatest (Fig. 4) (Kirchhoff et al. 2004). A recent extension of these measurements of kinetics is the repeated application of light dark intervals to leaves in a state of change. This general approach has been termed DIRK (Sacksteder & Kramer 2000). The analysis of the transients recorded during the DIRK procedure allows the changes in kinetics underlying the response to be resolved. Problems with measurement of P700 Two problems arise with the measurement of P700 oxidation state using light-induced absorbance changes. The first results from the overlap of absorbance changes caused by plastocyanin with those of P700 in the nm spectral region. The second is the possible loss of PSI efficiency because of a shortage of electron acceptors; this loss of efficiency will not be detected by techniques that use the relative amount of P700 + to quantify F PSI as shown in Eqn 3. The overlap between absorbance changes of plastocyanin and P700 is strong; and at around 820 nm, about 30% of the total absorbance change would be expected to derive from plastocyanin, with the proportion varying with wavelength (Klughammer & Schreiber 1991; Kirchhoff et al. 2004). This wavelength dependency has been exploited in deconvolution procedures to separate the contributions from plastocyanin and P700 to absorbance changes in vivo and in vitro (Kirchhoff et al. 2004). Under conditions where P700 and plastocyanin reach equilibrium, the absorbance changes reflect the expected electrochemical equilibrium between P700 and plastocyanin. Upon switching off the actinic light, first P700 is reduced, followed by plastocyanin and cytochrome f (Klughammer & Schreiber 1991; Sacksteder & Kramer 2000; Kirchhoff et al. 2004). If equilibrium is achieved on the time-scale of the normal turnover of the cytochrome b 6f complex, and the equilibrium constant for sharing electrons is constant, a simple model can be used to allow measurements of the absorbance change around 820 nm to yield accurate information about the electron flux through the cytochrome b 6f complex and P700 (Sacksteder & Kramer 2000). However, in many cases the apparent equilibrium constant changes suggesting partial disequilibrium among the electron carriers (Sacksteder & Kramer 2000; Kirchhoff et al. 2004). In this case, it is necessary to consider the kinetics of reduction of all of the carriers. It is clear in many cases that the estimates of F PSI based on absorbance changes around 820 nm correlate well with other estimates of leaf photosynthetic efficiency (Harbinson, Genty & Baker 1989; Genty & Harbinson 1996). This contradiction can be resolved in two ways. Firstly, in systems with rapid electron transport, there appears to be a restriction in the equilibration between plastocyanin and P700, and the apparent equilibrium constant is reduced from a value in the range expected from the redox potentials to one in range of 12 4 (Kirchhoff et al. 2004), dependent on the rate constant for P700 + reduction; the lower value is reached with rate constants of over 100 s -1, which would be normal for plants with a high rate of CO 2 fixation, such as crop plants. Consequently, in vivo absorbance changes caused by P700 and plastocyanin vary in parallel. Secondly, leaves and other photosynthetic systems are usually optically dense, for example, the average absorption of a leaf is around 84% which results in large gradients of irradiance through the system. Along such gradients, there will be a continuum of photochemically generated couples of P700 + /P700 and plastocyanin + / plastocyanin. Even if these couples are at equilibrium, the effect of the irradiance gradient is such that when the apparent equilibrium between P700 and plastocyanin is calculated from measurements that integrate over all these couples, it tends to unity as the absorbance approaches infinity (Harbinson & van Vliet 1994).This would also result in parallel changes in absorbance caused by plastocyanin and P700 oxidation. To verify that P700 oxidation is possible and not limited by a shortage of electron acceptors, it is necessary to examine the oxidizability of the P700 pool using a saturating light-pulse technique similar to that used to measure PSII efficiency (Klughammer & Schreiber 1994). Results from this technique verify that under most conditions, there is no shortage of PSI acceptors. Only during photosynthetic induction (Harbinson & Hedley 1993; Klughammer & Schreiber 1994), low-carbon dioxide concentrations under non-respiratory conditions (Genty & Harbinson 1996), or when the pool of PSI acceptors has been diminished (Holtgrefe et al. 2003) does the pool of acceptors appear to limit P700 oxidation. Under these conditions, the measurement of F PSI needs to take account of the decrease in efficiency caused not only by P700 + but also to those PSI reaction centres where photochemistry is impossible because of a shortage of acceptors. This can be done using the saturating flash technique to determine the proportion of PSI that is non-oxidizable and combining this with the conventional estimate of F PSI based on the proportion of P A possible source of error with the saturating pulse technique is

10 1116 N. R. Baker et al. that the multiple turnovers of PSI induced by the flash could close some open reaction centres by over-reducing their acceptor pools. There is, therefore, the risk of overestimating the degree of reaction centre closure using this technique, especially at low irradiances where the degree of reduction of high-potential PSI donors (plastocyanin and cytochrome f) will be high and the metabolic activity of the stroma low. PROTON FLUXES The proton circuit of photosynthesis plays a central role in energy transduction by the thylakoids, but is often given less attention than the associated electron transfer reactions. The light-driven fluxes of protons act not only to store energy for the synthesis of ATP, but also as a key regulatory component: activating the down-regulation of PSIIassociated antenna and governing electron transfer by controlling the oxidation of PQH 2 at the cytochrome b 6f complex. Generation of pmf by electron transfer-coupled reactions The light reactions drive the energy-requiring generation of an electrochemical potential difference, or pmf, across the thylakoid membrane. To accomplish this, proton translocation is coupled to (i.e. powered by) electron transfer reactions at four key points, making up a Mitchellian chemiosmotic energy storage system. As shown in Fig. 1, protons are released at two sites, the oxygen-evolving complex (OEC) of PSII and the plastoquinol-oxidizing (Q o) site of the cytochrome b 6f complex, and are taken up at the plastoquinone reductase site of PSII (Q B site) and the plastoquinone reductase site of the cytochrome b 6f complex (usually termed the Q i site, but sometimes the Q n site). In the OEC, four electrons are extracted from a pair of water molecules, resulting in the generation of one molecule of O 2 and the release of four protons into the lumenal space. The electrons extracted from water are transferred across the membrane through a chain of redox carriers within the PSII complex. The accumulation of two electrons (i.e. after two photochemical excitations of P680) on a PQ bound at the Q B site of PSII results in the uptake of two protons from the stromal side of the membrane, followed by the release of a neutral plastoquinol (PQH 2) into the thylakoid membrane. The PQH 2 is free to diffuse around, with some restrictions (Kirchhoff et al. 2004), until it is oxidized at the Q o site via turnover of the cytochrome b 6f complex via the Q-cycle. The Q-cycle is catalysed by the cytochrome b 6f complex. The presentation of two high-resolution structures (Kurisu et al. 2003; Stroebel et al. 2003) indicated that the overall structure of the cytochrome b 6f complex was similar to that of the related mitochondrial and bacterial cytochrome bc 1 complexes, strongly supporting the operation of very similar mechanisms in both systems. Most importantly, the structure is broadly consistent with the Q-cycle mechanism previously proposed by a number of laboratories (Rich 2004; Cape, Bowman & Kramer 2006). The key step in the Q-cycle is the bifurcated oxidation of PQH 2 into two distinct chains of electron carriers. In most Q-cycle models, one electron from the PQH 2 bound to the Q o site is transferred to the high-potential chain consisting of the Rieske FeS cluster and cytochrome f, followed in chloroplasts by the mobile carrier plastocyanin (Cape et al. 2006). This process leaves a reactive semiquinone radical in the Q o site, which reduces the low potential chain comprising of two cytochrome b hemes and the newly discovered c-type heme, heme c i (Kurisu et al. 2003; Stroebel et al. 2003). When two electrons have accumulated in the low potential chain, PQ is reduced to PQH 2 at the Q i site of the cytochrome b 6f complex with uptake of protons from the stroma. The Q-cycle appears to operate continuously in vivo, with very low rates of side reactions (Rich 1988, 2004; Kramer & Crofts 1993; Sacksteder et al. 2000). Consequently, it has been suggested that the overall proton pumping stoichiometry (H + /e - ratio) for LEF is 3, i.e. one proton released into the lumen at the level of water oxidation and two released during PQH 2 oxidation at the Q o site of the cytochrome b 6f complex (Allen 2003; Kramer et al. 2004a), rather than the value of 2 expected in the absence of a Q-cycle. The role of the pmf in ATP synthesis The mechanism of ATP synthesis driven by proton transfer through the ATP synthases has been extensively reviewed (Junge 1999; Stock, Leslie & Walker 1999; Seelert et al. 2000; Stock et al. 2000; Herbert 2002). High-resolution structural information and a likely molecular mechanism have allowed precise (but putative because they are based on a presumed mechanism) estimates of the H + /ATP ratio for steady-state ATP synthesis (Junge 1999; Stock et al. 2000; Ort & Baker 2002; Allen 2003). A full rotation of the g-subunit within the (C)F 1 a/b trimer of the coupling factor should form three molecules ATP from ADP and phosphate. The transfer of a single proton is thought to rotate the a subunit assemblage by a single c-subunit of the (C)F 0 ring. It follows that the number of c-subunits in the ring determines the H + /ATP ratio. This ratio varies depending upon species, but was found by atomic force microscopy to be 14 c-subunits/ring in chloroplasts (Seelert et al. 2000). Overall, this implies an H + /ATP ratio of 4.66, and taken together with the H + /e - stoichiometry of 3, indicates an ATP/NADPH ratio of ca. 1.3 for LEF (Allen 2003; Kramer et al. 2004a). Consequently, this has generated a renewed interest in mechanisms that can alter this ratio. The role of the pmf in regulating light processing and electron transfer As discussed in the section on PSII, chloroplasts can downregulate the rate of excitation of P680 by developing