TY - JOUR
AU - Rämma, Heikko
AB - Abstract Sunflower ( Helianthus annuus L.) and tobacco ( Nicotiana tabacum L.) were grown in the laboratory and leaves were taken from field-grown birch trees ( Betula pendula Roth). Chlorophyll fluorescence, CO 2 uptake and O 2 evolution were measured and electron transport rates were calculated, JC from the CO 2 uptake rate considering ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation, JO from the O 2 evolution rate, and JF from Chl fluorescence parameters. Mesophyll diffusion resistance, rmd , used for the calculation of JC , was determined such that the in vivo Rubisco kinetic curve with respect to the carboxylation site CO 2 concentration became a rectangular hyperbola with Km (CO 2 ) of 10 μM at 22.5°C. In sunflower, in the absence of external O 2 , JO = 1.07 JC when absorbed photon flux density (PAD) was varied, showing that the O 2 -independent components of the alternative electron flow to acceptors other than CO 2 made up 7% of JC . Under saturating light, JF , however, was 20–30% faster than JC , and JF − JC depended little on CO 2 and O 2 concentrations. The inter-relationship between JF − JC and non-photochemical quenching (NPQ) was variable, dependent on the CO 2 concentration. We conclude that the relatively fast electron flow JF − JC appearing at light saturation of photosynthesis contains a minor component coupled with proton translocation, serving for nitrite, oxaloacetate and oxygen reduction, and a major component that is mostly cyclic electron transport around PSII. The rate of the PSII cycle is sufficient to release the excess excitation pressure on PSII significantly. Although the O 2 -dependent Mehler-type alternative electron flow appeared to be under the detection threshold, its importance is discussed considering the documented enhancement of photosynthesis by oxygen. Introduction The discovery of 14 subunit IIIs in the ring structure of the CF 0 of the ATP synthase (Seelert et al. 2000 , Scheuring et al. 2001 ) raises the question of how the 3ATP/2NADPH ratio is guaranteed during photosynthesis. If the number of subunits determines the proton requirement per turn of the rotor, then 14H + /3ATP are consumed. In this case, the linear electron flow for CO 2 reduction is able to support the synthesis of only 2.57ATP/2NADPH, provided that the Q-cycle obligatorily operates in chloroplasts (Rich 1988 , Sacksteder et al. 2000 ). The proton deficiency of at least 17% must be covered by additional H + -coupled electron flow, such as cyclic electron flow around PSI or linear electron flow to alternative acceptors other than CO 2 , fine-controlled to match exactly the variable metabolic needs for non-photosynthetic ATP consumption and for the build-up of the lumenal proton concentration (Avenson et al. 2005 ). The low lumenal pH is the primary signal for control processes in photosynthetic electron transport, such as non-photochemical quenching (NPQ) of PSII excitation (Horton et al. 2000 , Niyogi et al. 2005 ) and down-regulation of cytochrome b 6 f turnover (Siggel 1974 , Rich and Bendall 1981 ). In this work, we investigated whether electron flow to acceptors other than CO 2 , usually termed the alternative electron flow, JAlt , can be the fine-tuning corrector of the ATP/NADPH ratio during photosynthesis. We also investigated whether JAlt can dissipate a part of excess light energy (Osmond and Grace 1995 , Kato et al. 2003 ). Three components of JAlt have been recognized: (i) electron flow supporting (mainly) nitrite reduction (Robinson 1988 , Robinson 1990 ); (ii) Mehler-type O 2 reduction at the acceptor side of PSI or from other low-potential electron carriers, followed by ascorbate peroxidase reaction (‘water–water cycle’, Asada 2000 ); and (iii) the ‘malate valve’ (Fridlyand et al. 1998 ). The latter pathway begins with the NADPH-linked reduction of oxaloacetate by malate dehydrogenase (MDH), followed by shuttling of malate from the chloroplast to the cytosol (Ebbighausen et al. 1987 , Scheibe et al. 2005 ). The rate of JAlt is usually measured as the difference between electron transport rates (ETRs) through PSII and through the carbon reduction/oxidation cycle (CROC). The PSII ETR, JF , is calculated from Chl fluorescence measurements, while the CROC rate, JC , is calculated from net CO 2 exchange measurements considering the known Rubisco kinetics with respect to CO 2 and O 2 (Laisk and Loreto 1996 , von Caemmerer 2000 ). Reliable values of the difference can be obtained only if the components JF and JC are measured with high accuracy. The reported values of JAlt are still contradictory. For example, in watermelon leaves, JAlt was undetectable at limiting absorbed photon flux densities (PADs), but increased at light saturation, forming about 30% of JF . Importantly, JAlt did not disappear when the ambient O 2 concentration was decreased to zero, but the O 2 -independent part of JAlt was 50–70% of the total. The O 2 -dependent part of JAlt followed the affinity of the ferredoxin and mono-dehydroascorbate reductase-catalyzed photoreduction of O 2 , while the O 2 -independent part of JAlt was suggested to reflect nitrite reduction (Miyake and Yokota 2000 ) or the PSII cycle (Miyake and Yokota 2001 ). Contrary to this, no clear evidence about the existence of JAlt was obtained from similar measurements on wild-type and Rubisco-deficient transgenic tobacco (Ruuska et al. 2000 ). The results of such measurements are dependent on the instruments used, considered correction factors (e.g. PSI fluorescence) and the interpretation of the fluorescence signal. For example, if NPQ of fluorescence varies rapidly during the measurement of the pulse-saturated Fm (Peterson and Havir 2004 ), then the calculated PSII electron transport may be slower and JAlt may be correspondingly smaller or zero. Thus, it is even not clear whether JAlt is present at any significant rate at all, not taking into account its internal fractionation between nitrite, oxaloacetate and oxygen reduction (Badger et al. 2000 ). In this work we investigated the alternative electron transport more closely under different experimental conditions, including low CO 2 and O 2 concentrations, where JAlt dominated in the photosynthetic electron flow and could be measured reliably. Our fluorescence measurements detected significant ETR through PSII under conditions where CO 2 and O 2 reduction were minimized by their low concentrations: then the simultaneously measured O 2 evolution rate was significantly slower than the fluorescence-detected PSII ETR. We concluded that fluorescence measurements detect significant cyclic electron flow around PSII, not accompanied by O 2 evolution in leaves, confirming the conclusion of Miyake and Yokota ( 2001 ), obtained on isolated thylakoids. To avoid confusion in terminology, we will not denote the difference between PSII and carbon reduction ETRs as JAlt , but use an empirical denotation JF − JC = JF−C , where JF denotes fluorescence-detected ETR and JC is the ETR accepted by CROC. We discuss that JF−C = JIICyc + JAlt , where the PSII cyclic electron flow, JIICyc , forms the major, and the alternative electron flow, JAlt , the minor part. Results Determining r md from the convexity of the Rubisco CO 2 response. The ETR JC for carbon reduction/oxygenation in CROC is critically dependent on the CO 2 and O 2 concentrations near the carboxylation/oxygenation sites of Rubisco (Equation 2 ). While the O 2 concentration changes relatively little, the carboxylation site CO 2 concentration depends on the gas phase diffusion resistance, rgw (including leaf boundary layer, stomatal and intercellular space resistance), and mesophyll diffusion resistance, rmd , for CO 2 transport in the liquid phase from cell walls to the carboxylation sites. The gas phase resistance was determined from transpiration measurements using traditional methods (Gaastra 1959 , Laisk and Oja 1998 ). We did not determine rmd using the method based on the consumption of electrons for ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation (Harley et al. 1992 , Loreto et al. 1994 , Laisk and Loreto 1996 ), since our aim was just the measurement of electron transport itself. Instead, we made use of the Rubisco kinetics, selecting an rmd value such that the in vivo kinetic curve of CO 2 fixation with respect to the calculated reaction site concentration, Cc , became the closest to the Michaelis–Menten-type hyperbola with Km (CO 2 ) of the value obtained from in vitro experiments. The fast response of the leaf CO 2 uptake measurement system made it possible to measure the Rubisco kinetic curve over a wide range of CO 2 concentrations, increasing the reliability of the obtained rmd values. The measurements were carried out at the external O 2 concentration of 20 mmol mol −1 in order to minimize photorespiration. At the external CO 2 concentration of 200 μmol mol −1 photosynthesis was still CO 2 limited, and RuBP accumulated, making a good starting position for the measurement of the kinetic curve. Transients to lower CO 2 concentrations did not induce changes in the RuBP level, representing steady-state points of the CO 2 response at the constant RuBP level ( Fig. 1 ). However, when the CO 2 concentration was increased, RuBP regeneration became rate limiting. In order to relate the measured kinetic curve to the initial RuBP level, CO 2 concentration was rapidly (within 0.5 s) increased to higher values and the initial CO 2 fixation rate was measured. The time course of the CO 2 fixation rate passed through an initial maximum and declined due to the decreasing RuBP concentration. The recorded CO 2 uptake rate was plotted vs. the integral of consumed RuBP, and the curve was extrapolated to zero RuBP consumption, with the aim of detecting the initial CO 2 uptake rate corresponding to the maximum RuBP pool (for details see Laisk et al. 2002 ). For every measured initial CO 2 fixation rate the Rubisco site CO 2 concentration, Cc , was calculated, using the measured gas phase diffusion resistance rgw , but setting the liquid phase diffusion resistance, rmd , such that the kinetic curve became the closest to the rectangular hyperbola with Km (CO 2 ) of 10 μM ( Fig. 1 ). The Km (CO 2 ) value was chosen according to in vitro measurements by Yokota and Kitaoka ( 1985 ). Fig. 1 View largeDownload slide Estimating the mesophyll diffusion resistance rmd from Rubisco kinetics in vivo. The RuBP pool was maximized in the steady state at 200 μmol CO 2 mol −1 and 20 mmol O 2  mol −1 at a PAD of 760 μmol quanta m −2  s −1 in a sunflower leaf at 22.5°C (filled data points). Then the CO 2 concentration was instantly changed to other values and the initial CO 2 fixation rate was measured (for details see Laisk et al. 2002 ). The Rubisco site CO 2 concentration Cc was calculated for the initial rate assuming different rmd values (indicated in the panel). The rmd value of 0.015 s mm −1 was selected since it resulted in the kinetic curve closest to a Michaelis–Menten-type rectangular hyperbola with Km (CO 2 ) of 10 μM (residuals between the calculated and measured data points shown in the panel below and in ΣR 2 ). gc is the carboxylation conductance (initial slope of the Rubisco kinetic curve), mm s −1 . Fig. 1 View largeDownload slide Estimating the mesophyll diffusion resistance rmd from Rubisco kinetics in vivo. The RuBP pool was maximized in the steady state at 200 μmol CO 2 mol −1 and 20 mmol O 2  mol −1 at a PAD of 760 μmol quanta m −2  s −1 in a sunflower leaf at 22.5°C (filled data points). Then the CO 2 concentration was instantly changed to other values and the initial CO 2 fixation rate was measured (for details see Laisk et al. 2002 ). The Rubisco site CO 2 concentration Cc was calculated for the initial rate assuming different rmd values (indicated in the panel). The rmd value of 0.015 s mm −1 was selected since it resulted in the kinetic curve closest to a Michaelis–Menten-type rectangular hyperbola with Km (CO 2 ) of 10 μM (residuals between the calculated and measured data points shown in the panel below and in ΣR 2 ). gc is the carboxylation conductance (initial slope of the Rubisco kinetic curve), mm s −1 . Determining J Alt from simultaneous measurements of CO 2 uptake and O 2 evolution As the first step, we measured the rate of the O 2 -independent components of JAlt under conditions where oxygen reduction was impossible. Simultaneous measurements of CO 2 uptake and O 2 evolution were carried out under very low external O 2 concentrations of 20–100 μmol mol −1 , insufficient for any significant O 2 reduction by the photosynthetic electron transport chain. At this O 2 concentration, the reduction of oxaloacetate plus nitrite along with 3-phosphoglyceric acid (PGA) was expected to cause somewhat faster O 2 evolution than CO 2 fixation. In Fig. 2 , the ETR calculated from O 2 evolution ( JO , Equation 4 ) is plotted against the ETR for CO 2 reduction ( JC , Equation 2 , [O 2 ] = 0) using data obtained from the measurement of light and CO 2 response curves on a sunflower leaf. Over the whole light response curve, JO and JC were proportional, but JO was 7% faster than JC . When photosynthesis was decreased by progressively stronger CO 2 limitation, the excess O 2 evolution remained at about a constant rate. The result shows that the non-Mehler components of JAlt , dominating in the difference JO−C = JO − JC , are rather small, but detectable from the difference between the O 2 evolution and CO 2 fixation rates. Fig. 2 View largeDownload slide Interdependence between electron transport rates calculated from the parallel measurements of CO 2 uptake, JC , and O 2 evolution, JO , in a sunflower leaf at the external O 2 concentration of 10–50 μmol mol −1 . For the measurement of light response, PAD was decreased stepwise from 1,300 μmol quanta m −2  s −1 to zero at the ambient CO 2 concentration of 300 μmol mol −1 (open symbols). For the measurement of CO 2 response, the CO 2 concentration was decreased stepwise from 300 μmol mol −1 to zero at a PAD of 1,300 μmol quanta m −2  s −1 (filled symbols). Fig. 2 View largeDownload slide Interdependence between electron transport rates calculated from the parallel measurements of CO 2 uptake, JC , and O 2 evolution, JO , in a sunflower leaf at the external O 2 concentration of 10–50 μmol mol −1 . For the measurement of light response, PAD was decreased stepwise from 1,300 μmol quanta m −2  s −1 to zero at the ambient CO 2 concentration of 300 μmol mol −1 (open symbols). For the measurement of CO 2 response, the CO 2 concentration was decreased stepwise from 300 μmol mol −1 to zero at a PAD of 1,300 μmol quanta m −2  s −1 (filled symbols). Calculating electron transport from Chl fluorescence measurements Equation 6 for the calculation of PSII electron transport assumes the reaction center–antenna equilibrium of excitation (RRP; reversed radical pair model). A critical test for this model is the slope of unity of the interdependence between 1/ F0 and 1/ Fm (Dau 1994 ). In Fig. 3 we have normalized fluorescence to Fmd , the maximum dark-adapted (pre-dawn) Fm , in order to cancel out the sensitivity of the measurement system. As a result, the point of intercept on the ordinate scales to kP0 , the relative rate constant for photochemistry (Laisk et al. 1997 , Peterson et al. 2001 ). Our results in Fig. 3 are in accordance with the RRP model, encouraging us to apply Equation 6 for the calculation of JF (similar results were obtained earlier with sunflower; Peterson et al. 2001 ). The value of kP0 of 6.3 is the same as that obtained in other birch leaves: the rate constant for photochemistry is 6–6.5 times faster than the rate constant for the basic decay of excitation, kf + kd (Eichelmann et al. 2004a , Eichelmann et al. 2004b ). We also noticed a small relative increase in F0 during the dark–light induction of photosynthesis (not shown) and under the lowest CO 2 concentrations in 20 mmol O 2 mol −1 (rightmost filled diamonds in Fig. 3 ), which could indicate the slight increase of PSI fluorescence under the strict limitation of electron transport (e.g. from 19 to 21% of F0 for the data in Fig. 3 ). Fig. 3 View largeDownload slide Interdependence between Fmd / Fm and Fmd / F0 during the variation of the qE -type non-photochemical quenching in a birch leaf. Non-photochemical quenching was varied by changing the light intensity, CO 2 and O 2 concentrations. The dotted line is drawn with a slope of unity. Measurements were carried out in the presence of 210 mmol O 2  mol −1 and 360 μmol CO 2  mol −1 by changing PAD (open squares) or changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 (filled squares) or in the presence of 20 mmol O 2  mol −1 and 360 μmol CO 2  mol −1 by changing PAD (open diamonds) or changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 (filled diamonds). Fig. 3 View largeDownload slide Interdependence between Fmd / Fm and Fmd / F0 during the variation of the qE -type non-photochemical quenching in a birch leaf. Non-photochemical quenching was varied by changing the light intensity, CO 2 and O 2 concentrations. The dotted line is drawn with a slope of unity. Measurements were carried out in the presence of 210 mmol O 2  mol −1 and 360 μmol CO 2  mol −1 by changing PAD (open squares) or changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 (filled squares) or in the presence of 20 mmol O 2  mol −1 and 360 μmol CO 2  mol −1 by changing PAD (open diamonds) or changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 (filled diamonds). A critical parameter of Equation 6 is the partitioning of excitation to PSII (relative optical cross-section of PSII antenna), aII . This parameter was determined from the measurements of the photosynthetic ETR at strictly limiting PADs. In order to consider excitation losses indicated by Chl fluorescence, the PSII quantum yield parameter YII = ( Fm − F )/ Fm was measured in parallel with the quantum yield of carbon reduction YC (Equation 3 ). The plot of the quantum yield of electron transport supporting RuBP carboxylation plus oxygenation, YC , against the quantum yield of PSII, YII , resulted in a curvilinear response with the maximum values at limiting PADs ( Fig. 4 ). The partitioning of excitation to PSII antenna, aII , was found by extrapolating the graph to the PSII efficiency of 1.0 (this considers the loss of quanta indicated by fluorescence, Eichelmann and Laisk 2000 ). The resulting aII = 0.42 in the experiment of Fig. 4 with a sunflower leaf, independent of O 2 concentration. Considering that the actual ETR through PSII was actually faster than JC by JO−C , the aII value was increased by 7%, resulting in aII = 0.45. In principle it would have been correct to determine aII not from the CO 2 uptake, applying the 7% correction, but directly from the O 2 evolution measurements, but exposure under the very low O 2 concentration, technically inevitable for the O 2 evolution measurements, tended slowly to inhibit the photosynthetic machinery. Fig. 4 View largeDownload slide Determining the partitioning of excitation to PSII, aII . CO 2 uptake and Chl fluorescence were measured in a sunflower leaf under different light intensities and CO 2 and O 2 concentrations. The quantum yield of CO 2 reduction (Equations 2 and 3 ) is plotted against the quantum yield of PSII electron transport YII = 1 − F / Fm . The extrapolation of the low-light data points (the highest yields) to YII = 1.0 results in the partitioning coefficient of excitation to PSII antenna, aII (solid line). The dashed line considers the 7% correction for the alternative electron flow determined from JO−C . Open squares, measurements in the presence of 210 mmol O 2  mol −1 changing PAD at 360 μmol CO 2  mol −1 ; filled squares, changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 ; open diamonds, measurements in the presence of 20 mmol O 2  mol −1 changing PAD at 360 μmol CO 2  mol −1 ; filled diamonds, changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 . The dotted line indicates the partitioning coefficient (PSII antenna cross-section) at light saturation, the photosynthetic efficiency decreased due to the PSII cycle. Fig. 4 View largeDownload slide Determining the partitioning of excitation to PSII, aII . CO 2 uptake and Chl fluorescence were measured in a sunflower leaf under different light intensities and CO 2 and O 2 concentrations. The quantum yield of CO 2 reduction (Equations 2 and 3 ) is plotted against the quantum yield of PSII electron transport YII = 1 − F / Fm . The extrapolation of the low-light data points (the highest yields) to YII = 1.0 results in the partitioning coefficient of excitation to PSII antenna, aII (solid line). The dashed line considers the 7% correction for the alternative electron flow determined from JO−C . Open squares, measurements in the presence of 210 mmol O 2  mol −1 changing PAD at 360 μmol CO 2  mol −1 ; filled squares, changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 ; open diamonds, measurements in the presence of 20 mmol O 2  mol −1 changing PAD at 360 μmol CO 2  mol −1 ; filled diamonds, changing CO 2 concentration under a PAD of 720 μmol quanta mol −1 . The dotted line indicates the partitioning coefficient (PSII antenna cross-section) at light saturation, the photosynthetic efficiency decreased due to the PSII cycle. Dependence of J F−C on light intensity, CO 2 and O 2 concentrations Using the above-determined aII in Equation 6 , the resulting PSII electron transport rate, JF , was, by definition, 7% faster than the electron transport supporting RuBP carboxylation and oxygenation, JC , at limiting PADs ( Fig. 5 ). However, at high PADs, JF continued to grow, saturating at a significantly higher rate than JC . Thus, the difference JF−C , only about 7% of the linear electron flow at limiting light, increased significantly as soon as the light intensity became saturating for photosynthesis. Fig. 5 View largeDownload slide Light response curves of PSII electron transport JF (open symbols) and electron transport for carbon reduction JC (filled symbols) at 360 μmol CO 2  mol −1 and 210 (squares) or 20 mmol O 2  mol −1 (diamonds) in the sunflower leaf of Fig. 4 . Fig. 5 View largeDownload slide Light response curves of PSII electron transport JF (open symbols) and electron transport for carbon reduction JC (filled symbols) at 360 μmol CO 2  mol −1 and 210 (squares) or 20 mmol O 2  mol −1 (diamonds) in the sunflower leaf of Fig. 4 . The results of measurements at different PADs and CO 2 and O 2 concentrations are summarized in Fig. 6 , where JF is plotted vs. JC . Both variables were, first, decreased by decreasing PAD, then returned to the initial, saturating PAD and decreased by decreasing the CO 2 concentration. At light limitation, JF changed 7% faster than JC , but at high PADs JF saturated at a rate significantly exceeding JC . The difference JF−C remained about constant independent of the CO 2 concentration at 210 mmol O 2  mol −1 , but decreased somewhat at the most strictly limiting concentrations of 50 and 0 μmol CO 2  mol −1 at 20 μmol O 2  mol −1 . In this sunflower leaf, JF−C was 40–60 μmol m −2  s −1 or about 30% of the whole ETR at saturating light, and no significant difference could be detected dependent on O 2 concentration between 210 and 20 μmol O 2  mol −1 . The decrease of JF−C at the lowest CO 2 concentrations at 20 mmol O 2  mol −1 was, rather, caused by the strict limitation of ETR, not by the low O 2 concentration. In similar experiments with tobacco leaves, even the very low O 2 concentration of <50 μmol mol −1 did not significantly influence JF−C , which was 10.2 ± 0.7, 11.4 ± 0.8 and 12.3 ± 1.1 μmol e −  m −2  s −1 at 210, 20 and <0.05 mmol O 2  mol −1 , respectively, measured as an average at two CO 2 concentrations of 200 and 360 μmol mol −1 at each O 2 concentration ( n = 8). Fig. 6 View largeDownload slide Dependence between the PSII electron transport calculated from Chl fluorescence parameters (Equation 6 ) and electron transport for carbon reduction (Equation 2 ) in the sunflower leaf of Fig. 4 . Light response curves were measured at 360 μmol CO 2  mol −1 and O 2 concentrations of 210 (open squares) and 20 mmol mol −1 (open diamonds) decreasing PAD from 720 to 0 μmol m −2  s −1 at the CO 2 concentration of 360 μmol mol −1 . CO 2 response curves were measured by decreasing the CO 2 concentration from 360 to 0 μmol mol −1 at a PAD of 720 μmol quanta m −2  s −1 (filled symbols). Dashed line, JF = JC . Fig. 6 View largeDownload slide Dependence between the PSII electron transport calculated from Chl fluorescence parameters (Equation 6 ) and electron transport for carbon reduction (Equation 2 ) in the sunflower leaf of Fig. 4 . Light response curves were measured at 360 μmol CO 2  mol −1 and O 2 concentrations of 210 (open squares) and 20 mmol mol −1 (open diamonds) decreasing PAD from 720 to 0 μmol m −2  s −1 at the CO 2 concentration of 360 μmol mol −1 . CO 2 response curves were measured by decreasing the CO 2 concentration from 360 to 0 μmol mol −1 at a PAD of 720 μmol quanta m −2  s −1 (filled symbols). Dashed line, JF = JC . Determining mesophyll diffusion resistance using the O 2 independence of J F−C The carboxylation site CO 2 concentration Cc , calculated dependent on the mesophyll diffusion resistance rmd (Equation 1 ), has an effect on the calculated JC only in the presence of oxygen (Equation 2 ). Assuming now that the Mehler-type O 2 reduction is much slower than RuBP oxygenation, this condition can be used for the calculation of an rmd value, such that JF−C becomes independent of the O 2 concentration. An example of this approach on a birch leaf is given in Fig. 7 . CO 2 response curves at ambient CO 2 concentrations, Ca , from 360 to 0 μmol mol −1 were measured at different O 2 concentrations from 500 to 20 mmol mol −1 . Electron transport supporting CROC, JC , was calculated assuming different mesophyll diffusion resistances from 0.02 to 0.08 s mm −1 . At rmd = 0.05 s mm −1 , JF−C became independent of the O 2 concentration. This rmd formed one-third of the total mesophyll resistance (sum of diffusion and carboxylation resistances), rm = rc + rmd  = 0.154 s mm −1 in this birch leaf at 20 mmol O 2  mol −1 . At higher O 2 concentrations, the relative importance of rmd was smaller, because the carboxylation resistance, rc , increased. Fig. 7 View largeDownload slide CO 2 responses of electron transport, through PSII, JF (ordinate), and for carbon reduction/oxidation, JC , (abscissa), at different O 2 concentrations in a birch leaf. The PAD was 650 μmol quanta m −2  s −1 , CO 2 concentration was decreased stepwise from 360 to 0 μmol mol −1 . O 2 concentrations are shown in (B). Data were calculated using different rmd values, as shown in the panels. rmd = 0.05 s mm −1 resulted in O 2 -independent JF−C . Fig. 7 View largeDownload slide CO 2 responses of electron transport, through PSII, JF (ordinate), and for carbon reduction/oxidation, JC , (abscissa), at different O 2 concentrations in a birch leaf. The PAD was 650 μmol quanta m −2  s −1 , CO 2 concentration was decreased stepwise from 360 to 0 μmol mol −1 . O 2 concentrations are shown in (B). Data were calculated using different rmd values, as shown in the panels. rmd = 0.05 s mm −1 resulted in O 2 -independent JF−C . Relationship between J F−C and non-photochemical quenching If JF−C is coupled with proton transport during photosynthesis, the ΔpH-dependent NPQ is expected to be functionally related to it. To investigate this, we measured light response curves of photosynthesis and NPQ at different ambient CO 2 concentrations at the O 2 concentration of 20 mmol O 2  mol −1 . From Fig. 8 it is evident that at any CO 2 concentration the light dependence of JF−C was similar to that in Figs. 5 and 6 , i.e. JF−C was slow at light limitation, but increased significantly as soon as light became saturating. Even the low O 2 concentration of 20 mmol mol −1 supported ETR of 10 μmol m −2  s −1 through CROC, though CO 2 was absent. At the low O 2 and CO 2 concentrations applied in this experiment, JF−C formed a significant proportion of the total electron flow, thus the calculated value could not be influenced by a possible wrong estimate of the reassimilation of photorespiratory CO 2 . Fig. 8 View largeDownload slide Interdependence between PSII electron transport determined from Chl fluorescence, JF , and electron transport for carbon reduction/oxidation, JC , in a sunflower leaf. Light response curves were measured at different CO 2 concentrations (shown at the data points) and an O 2 concentration of 20 mmol mol −1 . Dotted line, JF = JC . Fig. 8 View largeDownload slide Interdependence between PSII electron transport determined from Chl fluorescence, JF , and electron transport for carbon reduction/oxidation, JC , in a sunflower leaf. Light response curves were measured at different CO 2 concentrations (shown at the data points) and an O 2 concentration of 20 mmol mol −1 . Dotted line, JF = JC . An approximately linear correlation was found between NPQ and JF−C at the highest CO 2 concentration used ( Fig. 9 ), suggesting an apparent functional relationship between the two parameters. However, the slope of the NPQ vs. JF−C relationship was not constant, but increased the lower the CO 2 concentration, and, finally, at zero external CO 2 concentration the full-range NPQ was generated before any significant JF−C could be detected, whilst JF−C changed over the full range at the constant NPQ. This result may be interpreted to show that the difference JF−C contains two components. The component responsible for the build-up of the regulatory ΔpH is so small that is not detectable as the difference between JF and JC . The faster component contributing the major part of the difference JF−C is not related to NPQ. Fig. 9 View largeDownload slide Interdependence between non-photochemical quenching (Equation 5 ) and JF−C (data from the experiment of Fig. 8 ). Fig. 9 View largeDownload slide Interdependence between non-photochemical quenching (Equation 5 ) and JF−C (data from the experiment of Fig. 8 ). Discussion In this work, we used contemporary techniques for the simultaneous measurement of leaf CO 2 uptake, O 2 evolution and Chl fluorescence, with the aim of determining electron flow rates reflecting water splitting, JO , PSII electron transport, JF , and carbon reduction, JC . Under anaerobic conditions, eliminating the Mehler-type O 2 reduction, JO was 1.07 JC when both were varied by changing the light intensity. The result showed that nitrite reduction and the malate valve together formed about 7% of the light-saturated ETR. The malate valve system involves chloroplast MDH and the dicarboxylate (malate/oxaloacetate) translocator, enabling the transfer of light-generated reducing equivalents into the cytoplasm (Heber 1974 ). MDH is an allosterically regulated enzyme, activated by the ferredoxin–thioredoxin system and the reduction state of the NADP system (Scheibe and Jacquot 1983 , Scheibe 1987 ). Considering the properties of MDH, Ebbighausen et al. ( 1987 ) have proposed that the export of reducing equivalents out of chloroplasts via an oxaloacetate/malate exchange could play a significant role in vivo. On the enzyme activity basis, the rate of electron flow, other than PGA reduction, has been estimated to be 17% of the total flow through PSI, of which nitrite reduction accounts for 7.4%, O 2 reduction and the malate valve both contribute about 4.5%, and cyclic electron flow around PSI contributes 4% (Fridlyand et al. 1998 , Fridlyand and Scheibe 1999 ). In the absence of O 2 , this theory predicts the non-Mehler-type JAlt of 12% of the ETR, not far from our measured JO−C of 7%. To detect the Mehler-type O 2 reduction, we took special care regarding the correct measurement of JC at different O 2 concentrations, where the RuBP carboxylation/oxygenation ratio and reassimilation of photorespiratory CO 2 were critical. The Rubisco specificity factor Ks (Equation 2 ) was determined for every leaf from the O 2 dependence of the CO 2 photo-compensation point (Laisk et al. 2002 ). Mesophyll diffusion resistance, rmd , important for the correct calculation of the reassimilation of photorespiratory CO 2 , was determined from carboxylation kinetics, avoiding circular logic involved in the method of Harley et al. ( 1992 ), itself based on assumptions about JF and JC . The thus determined rmd values made up about 15–30% of the total mesophyll resistance ( rm = rc + rmd , the sum of carboxylation, rc , and diffusion resistance, rmd ), in accordance with our earlier reports (Eichelmann et al. 2004a , Eichelmann et al. 2004b , Laisk et al. 2005b ) and with other methods (Evans and Loreto 2000 , Warren 2006 ). Since the O 2 evolution rate and the related JO could not be measured at high O 2 concentrations in our flow-through gas exchange system, we determined the PSII electron transport rate from Chl fluorescence. Interdependence between 1/ Fm and 1/ F0 with the slope of 1 – the basic condition of the validity of Equation 6 for PSII electron transport (Dau 1994 ) – was found to be fulfilled, except for a slight disproportionate increase of F0 at strictly limiting CO 2 concentrations, probably indicating a small increase of PSI fluorescence under these conditions. The critical parameter of Equation 6 , the partitioning of excitation to PSII, aII , was set such that JII = 1.07 JC at low photon flux densities, applying the correction factor of 1.07, considering the measured JO−C . In earlier publications, we assumed that there was no alternative electron transport at limiting light (Laisk and Loreto 1996 ), resulting in aII values of 0.45–0.47 in different leaves and growth conditions (Laisk et al. 2002 , Eichelmann et al. 2004a , Eichelmann et al. 2004b , Eichelmann et al. 2005 , Laisk et al. 2005b ), though in tobacco a lower aII of 0.31 has been reported (Miyake and Yokota 2000 ). Applying the correction of 1.07 for the non-Mehler-type JAlt , the PSII cross-section in our above-cited publications would increase to 0.48–0.50. Thus, the frequent intuitive assumption that aII = 0.5 is actually correct. Our JF−C values obtained considering these methodical details were slow at limiting light and increased when photosynthesis became light saturated, in general agreement with earlier measurements (Loreto et al. 1994 , Laisk and Loreto 1996 , Miyake and Yokota 2000 , Makino et al. 2002 ). However, the oxygen-dependent Mehler-type alternative electron flow was very slow in leaves, actually below the detection limit with our techniques, and an unknown oxygen-independent component formed the major part of the light-saturated JF−C . This fast JF−C was best measured when electron transport was strictly acceptor limited (low or zero CO 2 and zero or 20 mmol O 2  mol −1 ). Under these conditions, JC was slow and JF−C comprised about 50% or more of the PSII electron transport. The whole leaf cross-section was uniformly light saturated, eliminating the possible effects of a light gradient in the leaf cross-section on the measurement of JF and JC (though such effects have been shown to be small anyway, Edwards and Baker 1993 ). Measurements at strictly limiting CO 2 and O 2 concentrations also eliminated the possibility that the PSII electron transport was overestimated at saturating light by using in Equation 6 the aII value determined at limiting light. It is known that the excitation partitioning to PSII may decrease due to the state transition-induced migration of the PSII light-harvesting complexes at light saturation (Andrews et al. 1993 , Allen and Pfannschmidt 2000 ). However, the state transitions involve no more than 10–15% of the PSII antenna, which is less than the observed JF−C of 25–30% of JF at the atmospheric CO 2 and O 2 concentrations. At the low CO 2 and O 2 concentrations, JF−C was even about 50% of the total ETR. Such a large state transition-induced decrease of PSII antenna has never been observed. Thus, the presence of fast PSII electron transport at the very low CO 2 and O 2 concentrations was clearly visible from the variable fluorescence and it could not be overestimated due to the neglected state transition-induced decrease of PSII antenna. If the whole JF−C , recorded at the low CO 2 and O 2 concentrations, were coupled with proton translocation, these protons had to be released from the lumen, despite the fact that ATP was not required for the carbon reduction cycle at that rate. A possible pathway could be the ‘slip’—the maintained flow of protons through ATP synthase at very low substrate concentrations (Feniouk et al. 2005 ). However, even if the ‘slip’ could be possible at the extremely low CO 2 and O 2 concentrations, it could not be possible under the atmospheric CO 2 and O 2 concentrations, where the photosynthetic ETR was close to the maximum and both substrates, ADP and P i , had to be readily available for the enzyme. The increase of JF−C could also reflect a sudden increase of ATP requirement in metabolism approaching light saturation. Starch synthesis requires 1ATP/6CO 2 , while for carbon reduction the demand is 3ATP/CO 2 . The corresponding JF−C providing ATP for starch synthesis could not exceed 5% of JC , but most probably was even less. Protein synthesis could not rapidly react to changes in light intensity, but the increase in JF−C was induced within seconds. Thus, the need for fast JF−C at light saturation could not be explained either by altering ATP requirements in the chloroplast or by the ‘slip’ of the ATP synthase. The conclusion from the above discussion is that the extra-fast JF−C present at light saturation of photosynthesis is not coupled with proton transport, at least for its major part. This conclusion is supported by the mutual dependence of JF−C and NPQ ( Fig. 9 ), indicating linear correlation with variable slope. Rather, JF−C contains two components. The H + -coupled component, responsible for the build-up of the regulatory ΔpH, is so small that it is not detectable as the difference between JF and JC . The faster component contributing the major part of JF−C is uncoupled from proton transport and is not the primary cause of NPQ. These facts taken together support the proposal that the fluorescence-detected extra ETR through PSII, present in the absence of O 2 and not coupled with transmembrane proton transport, is predominantly cyclic electron flow around PSII. The photoprotective role of electron cycling within PSII has been suggested from experiments on intact chloroplasts (Heber et al. 1979 , Whitmarsh and Pakrasi 1996 ). Experimentally, the PSII cycle has been detected from the increase in fluorescence accompanying the application of hydroxylamine, a competitive electron donor to the PSII oxidizing side (Horton and Lee 1983 ), or from the difference of the fluorescence-detected PSII quantum yield and the quantum yield of methyl viologen-catalyzed O 2 uptake in spinach thylakoid membranes—an experimental system eliminating PSI-catalyzed O 2 reduction (Rees and Horton 1990 ). In the latter experimental system, the PSII cycle has been shown to be active only in coupled thylakoids; in the presence of the protonophore nigericin it was activated only at pH below 5.5 (Miyake and Yokota 2001 ). The estimated rate of the PSII cycle was comparable with JF−C measured in watermelon leaves at light saturation (Miyake and Yokota 2000 ). The suggested pathway for the PSII cyclic electron flow involves the reduction of cytochrome b 559 by reduced Q A or Q B and donation of electrons to P680 + via Chl z (Whitmarsh and Pakrasi 1996 ). The activation of the cycle at the low lumenal pH may be caused by the weakening competition of the water splitting system for the reduction of P680 + . Though in different in vitro experiments the rate of the PSII cycle has generally been considered slow, in leaves its rate is sufficient to decrease the excitation pressure upon linear electron flow by 20–30% at light saturation ( Fig. 4 ). The PSII cycle and NPQ together are sufficient to dissipate the excess excitation even under rather severe conditions. The photoprotective role of the Mehler-type O 2 reduction is very limited indeed (Wiese et al. 1998 ). Although we could not detect any significant O 2 -dependent JAlt , the importance of O 2 reduction in photosynthesis is not in doubt. Without external oxygen the induction of photosynthesis is very slow in leaves (Laisk and Oja 1998 ) or impossible in isolated chloroplasts (Ziem-Hanck and Heber 1980 ). Under saturating CO 2 , when photorespiration is suppressed, atmospheric oxygen significantly enhances photosynthesis (Viil et al. 1972 , Viil et al. 1977 ). This shows the Mehler-type O 2 reduction, coupled with the water–water cycle, is active in leaves, but its rate is much slower than that of the PSII cycle. The PSII cycle eclipses the O 2 reduction, making it very difficult to detect it as the difference between the PSII electron flow, JF , and the PGA reduction rate, JC . However, the fact that such a small alternative ETR can significantly regulate the rate of photosynthesis indicates that the ATP/NADPH ratio supported by the linear electron flow for the PGA reduction is very close to the optimal value, needing only minor correction. A proposed role for JAlt is to adjust the ATP/NADPH synthesis ratio during photosynthesis. The discovery that in chloroplasts the F 0 complex of ATP synthase contains 14 subunits (Seelert et al. 2000 , Scheuring et al. 2001 ) suggests that the proton consumption per ATP is 14/3 = 4.67 instead of 4. In order to support the increased proton consumption by ATP synthase, JAlt must be at least 17% of the linear flow. Under anaerobic conditions, where photosynthesis was still fast and ATP synthase was functioning normally, we detected the non-Mehler-type JO−C = JAlt of only 7% of the linear flow. Of course, cyclic electron flow around PSI could fill the gap in proton transport, carrying at least 10% of protons. Cyclic electron flow has been detected around PSI (Makino et al. 2002 , Miyake et al. 2004 , Munekage et al. 2004 , Miyake et al. 2005 ), but, again, its rate was slow at light limitation and too fast at light saturation, not a constant proportion of the linear flow (Laisk et al. 2005a ). A doubt still remains that the proton requirement of 14H + /3ATP of the chloroplast ATP synthase is not complemented by the PSI cyclic electron flow either, leaving open the possibility that the 14 subunits in the CF 0 complex of the chloroplast ATP synthase does not necessarily mean that 14H + /S3ATP are required, but the requirement may still be close to 12H + /3ATP. However, more detailed measurements of the PSI cyclic electron flow are in progress in our laboratory, to strengthen this conclusion. Materials and Methods Plant material Sunflower ( Helianthus annuus L.) and tobacco ( Nicotiana tabacum L.) plants were grown in a fertilized peat–soil mixture in the laboratory under the photosynthetic photon flux density of 400–600 μmol quanta m −2  s −1 , 14 h/10 h day/night length and temperature of 22–25°C/16–18°C. Full-grown attached leaves of the laboratory-grown plants were used in experiments. Some of the experiments reported in this work were carried out on leaves taken from birch ( Betula pendula Roth) trees growing in open-top chambers under atmospheric CO 2 concentration (the control trees for the elevated CO 2 and O 3 experiments, Eichelmann et al. 2004a ). Leaves were cut from trees and, with their petiole in water, fit to the leaf chamber. Gas exchange measurements The properties of the two-channel fast-response leaf gas exchange measurement system (Fast-Est, Tartu, Estonia) have been described (Laisk et al. 2002 , Oja et al. 2003 ). In this system, the leaf was enclosed in a leaf chamber of 31 mm diameter and 3 mm height, at a gas flow rate of 0.5 mmol s −1 . With the aim of temperature stabilization, the upper epidermis of the leaf was sealed with starch paste against the window, flushed by thermostat water from the other side. Gas exchange could proceed only through the lower epidermis. In the experiments reported above, the water temperature was 22°C, and leaf temperature did not exceed 23°C at the maximum light intensity. The two-channel circuit allowed us rapidly to change CO 2 and O 2 concentrations and to calibrate the reference line and sensitivity of the LI 6251 CO 2 analyzer (LiCor, Lincoln, NE, USA) at each CO 2 and O 2 concentration. Transpiration was measured with a micro-psychrometer integrated in the gas system. Leaf gas phase diffusion resistance, as well as the dissolved mesophyll cell wall CO 2 concentration ( Cw , μM), were calculated from net CO 2 uptake, transpiration and leaf temperature (Laisk and Oja 1998 , Laisk et al. 2002 ). The carboxylation site CO 2 concentration, Cc , was calculated as   1 where A is net CO 2 uptake rate and rmd is mesophyll diffusion resistance. The latter was determined from the comparison of the leaf A vs. Cc curve with the in vitro kinetic curve of Rubisco, finding an rmd value such that the Km (CO 2 ) of the leaf A vs. Cc curve would become equal to 10 μM (see Results). Our concentration-based units for diffusion resistances may be converted into pressure-based or mole fraction-based units as follows: 1 s mm −1  = 2.80 Pa/(μmol m −2  s −1 ) = 27.3 (μmol mol −1 )/(μmol m −2  s −1 ). The rate of linear electron transport associated with photosynthetic carbon metabolism (PGA reduction rate), JC , was calculated considering RuBP carboxylation and oxygenation and the Kok effect of partial suppression of CO 2 evolution from the Krebs cycle in the light (Laisk and Loreto 1996 , Laisk and Oja 1998 , Peterson et al. 2001 ):   2 where A is the measured net rate of CO 2 assimilation, RK is the rate of Krebs cycle respiration in the light, Ks is the CO 2 /O 2 specificity factor for Rubisco, and Cc and Oc are the dissolved CO 2 and O 2 concentrations (μM) at the carboxylation site. Near the CO 2 compensation point Equation 2 becomes undefined, then we used Equation 2a , containing carboxylation conductance (the slope of the Rubisco kinetic curve) gc = dA / dCc at the CO 2 compensation point   2a Parameters of Equations 2 and 2a were determined for each leaf as described in Laisk et al. ( 2002 ). The corresponding quantum yield of carbon reduction was calculated as   3 where I is the photosynthetically active PAD. Oxygen evolution was measured in the same flow-through system at a very low background O 2 concentration of 10–50 μmol mol −1 (dependent on the photosynthetic rate) using a Zr-based O 2 analyzer Ametek S-3A (Thermox, Pittsburgh, PA, USA). The oxygen analyzer was calibrated by air flow through the same capillary which served for the calibration of the CO 2 analyzer by CO 2 flow. Using this method, only the ratio of viscosities of air and CO 2 remained the scaling factor between CO 2 and O 2 measurements, plus the considered small difference in the ‘slip’ radius for CO 2 and air (Oja and Laisk 2000 ). The reference, zero O 2 evolution, was measured by darkening the leaf (mitochondrial O 2 uptake was negligible at the low O 2 concentration). The ETR, JO , supporting the measured O 2 evolution rate, AO , was calculated as   4 Chl fluorescence measurements The leaf was uniformly illuminated through special fiber optics (Fast-Est, Tartu, Estonia), integrating illumination from three sources (white light, far-red light and saturation pulses of 10,000 μmol quanta m −2  s −1 ). Fibers for fluorescence measurements were systematically arranged between the illumination fibers, covering a 10 × 20 mm section in the leaf chamber. The arrangement of fibers perpendicularly to the surface at a distance of 15 mm from the leaf guaranteed a high signal/noise ratio and well defined geometry of fluorescence measurements, avoiding mutual shading of illuminating and measuring fibers. Chl fluorescence was measured with a PAM-101 and ED-101 emitter-detector (H. Walz, Effeltrich, Germany). The recorded Chl fluorescence yield values were corrected for (i) the residual (offset) response of the detector to the emitter light-emitting diode, caused by reflections from surfaces in the system (cross-sensitivity); (ii) the partial saturation of the ED-101 detector in the strong saturation pulse light; (iii) unsaturation of fluorescence in the saturation pulse; and (iv) PSI fluorescence (Peterson et al. 2001 ). The latter correction was 18–22% of F0 with the short-pass filter in ED-101, cutting beyond 760 nm in order to avoid interference between Chl fluorescence and 810 nm transmission measurements (not reported here). The relative rate constant for non-photochemical excitation quenching, kN , (Laisk et al. 1997 ), also termed NPQ (Bilger and Björkman 1990 ), was calculated as:   5 where Fmd is the completely dark-adapted (pre-dawn) pulse-saturated fluorescence yield Fm . 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TI - Photosystem II Cycle and Alternative Electron Flow in Leaves
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcj070
DA - 2006-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/photosystem-ii-cycle-and-alternative-electron-flow-in-leaves-uIiC89rToV
SP - 972
EP - 983
VL - 47
IS - 7
DP - DeepDyve
ER -