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, A0 (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 A0, the electron is transferred to one of a pair of A1 (phylloquinone), Fx, FA and FB (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 b6f complex. There are two sources for the electrons that reduce the cytochrome b6f 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 C3, photosynthetic organisms whose main photosynthetic activity is CO2 assimilation and/or photorespiratory O2 reduction, PSII is the predominant source of electrons that ultimately reduce P700. A high yield of PSI-driven CEF1 in such plants would be inconsistent with their high yield of CO2 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 C4 plants, organisms with more flexible metabolism (e.g. green algae), and possibly, stressed C3 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 O2 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 P700+. 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 vivo is monitored by means of a light-induced absorbance change, usually in the range 800–850 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 (P7000) is a measure of the ΦPSI, and is calculated from:
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% O2 with CO2 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 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 0.05–0.10 at PPFDs below 100 µmol m−2 s−1 because of reduction of QA (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 ΦPSI 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 ΦPSI (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.
Figure 4. A typical relationship between the quantum efficiency for electron transport by PSI (ΦPSI) 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. ΦPSI 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).
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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 ΦPSI to light intensity varies between leaves and also depends on the environmental and physiological conditions of the leaf at the time of measurement, for example, temperature, drought stress, leaf age, source/sink balance, CO2 and O2 concentration (Harbinson, Genty & Foyer 1990; Peterson 1991; Harbinson 1994; Laisk & Oja 1994). Decreases in ΦPSI at constant irradiance will also be produced by factors that decrease photosynthesis, such as decreasing CO2 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 b6f 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 b6f 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 b6f 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 b6f complex (Heber, Neimanis & Dietz 1988). In contrast, electron transport at low irradiance is more sensitive to inhibition at QB than it is at high irradiance (Heber et al. 1988). NPQ of PSII will diminish the rate of reduction of the QA pool, and will increase with increasing irradiance above the region of light limitation of photosynthesis. By analogy with the effect of inhibition at QB 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, ΦPSI can be used to estimate the electron flux through PSI (ETRPSI, also often termed JPSI):
where I is the incident PPFD on the leaf, Aleaf is the spectral leaf absorptance and fractionPSI is the fraction of the absorbed irradiance that is trapped by PSI complexes. It is difficult to determine fractionPSI 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 ΦPSI. 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 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 CO2 concentrations below 100 ppm when the O2 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 b6f complex rather than the acceptor side, even when metabolic demand for reductant is low. The rate of electron transport from the PQH2 pool to the cytochrome b6f 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 long-term control is caused by changes in the amount of the cytochrome b6f 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 ΔA820 absorbance change (Fig. 5). This decay, which has a half-time of 3–4 ms or greater, reflects the rate-limiting supply of reductant passing from PQH2 via the cytochrome b6f complex and plastocyanin to P700+. The rate constant for this supply of reductant to P700+, ke, 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 ke is that it is absolute, not relative like ΦPSI. This allows comparisons to be made between leaves and for the basis of changes in the relationship between ΦPSI and PPFD relationship to be analysed in terms of changes in ke (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 b6f 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 b6f 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.
Figure 5. Typical decay kinetics of the absorption change at 820 nm (ΔA820) 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 PQH2 and the cytochrome b6f 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.
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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 800–850 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 ΦPSI 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 b6f 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 b6f 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 ΦPSI 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 64–312 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 CO2 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 ΦPSI 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 ΦPSI based on the proportion of P700+. A possible source of error with the saturating pulse technique is 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.