TY - JOUR
AU - Chow, Wah, Soon
AB - Abstract Photosynthetic induction, a gradual increase in photosynthetic rate on a transition from darkness or low light to high light, has ecological significance, impact on biomass accumulation in fluctuating light and relevance to photoprotection in strong light. However, the experimental quantification of the component electron fluxes in and around both photosystems during induction has been rare. Combining optimized chlorophyll fluorescence, the redox kinetics of P700 [primary electron donor in Photosystem I (PSI)] and membrane inlet mass spectrometry in the absence/presence of inhibitors/mediator, we partially estimated the components of electron fluxes in spinach leaf disks on transition from darkness to 1,000 µmol photons·m−2·s−1 for up to 10 min, obtaining the following findings: (i) the partitioning of energy between both photosystems did not change noticeably; (ii) in Photosystem II (PSII), the combined cyclic electron flow (CEF2) and charge recombination (CR2) to the ground state decreased gradually toward 0 in steady state; (iii) oxygen reduction by electrons from PSII, partly bypassing PSI, was small but measurable; (iv) cyclic electron flow around PSI (CEF1) peaked before becoming somewhat steady; (v) peak magnitudes of some of the electron fluxes, all probably photoprotective, were in the descending order: CEF1 > CEF2 + CR2 > chloroplast O2 uptake; and (vi) the chloroplast NADH dehydrogenase-like complex appeared to aid the antimycin A-sensitive CEF1. The results are important for fine-tuning in silico simulation of in vivo photosynthetic electron transport processes; such simulation is, in turn, necessary to probe partial processes in a complex network of interactions in response to environmental changes. Introduction On a transition from darkness or low light to high light, photosynthesis in higher plants usually reaches a maximum rate only after an induction period of minutes. Such a transition in nature can occur, e.g. when understorey plants are exposed to sunflecks, or when prolonged cloud cover is followed by sunny weather. The potential ecological significance of sunflecks was recognized about a century ago (Osterhout and Haas 1918, Allee 1926). The slow transition from shade or low light to full sunlight is also a significant determinant of carbon gain in plants in the field. For example, slow induction of photosynthesis on transition from shade to sun may cost at least 20% of wheat productivity (Taylor and Long 2017), whereas accelerating the rate of relaxation of nonphotochemical quenching (NPQ) on transition from high light to shade can increase leaf carbon dioxide uptake and plant dry matter productivity by about 15% in fluctuating light (Kromdijk et al. 2016). The main mechanisms that regulate photosynthetic induction have been studied extensively (Walker 1973, Pearcy 1990, Pearcy and Way 2012, Kaiser et al. 2015). A major factor that underlies photosynthetic induction is the gradual accumulation of metabolites during the autocatalytic action of the Calvin–Benson cycle (Walker 1973, Prinsley and Leegood 1986), whereas stomatal responses can also play a part, especially later in the induction phase (Xu et al. 1992, Deans et al. 2019). In addition, faster photosynthetic induction can be achieved by increased rates of Rubisco activation (Hammond et al. 1998, Soleh et al. 2016) and, in tobacco, by expressing cyanobacterial flavodiiron proteins in chloroplasts (Gómez et al. 2018). Also in tobacco, a defect in the gene that codes for the chloroplast NADH dehydrogenase-like complex (NDH) delays photosynthetic induction at high CO2 concentration (Martin et al. 2015). In C4 leaves, the carbon metabolism is more complex; the initial phase of CO2 uptake during induction could be associated with both the rearrangement of carbon distribution in both the C3 and C4 cycles and the activation of key C4 enzymes (Furbank and Walker 1985). Interestingly, in leaves of tomato plants grown under various mixtures of red and blue light, the time course of photosynthetic induction was not different despite differing photosynthetic properties such as CO2 assimilation rate (Zhang et al. 2019). By contrast, across fern, gymnosperm and angiosperm species, stomatal opening time is positively correlated to the maximum CO2 carboxylation rate at high irradiance (Deans et al. 2019). Although the effects of carbon metabolism during induction have been extensively investigated, there are relatively few studies of electron transport events in the two photosystems during induction. Kaiser et al. (2017) reported induction of the overall photochemical yield, Y(II), of Photosystem II (PSII) and NPQ from induction time 2 min onward; they showed that changes in Y(II) during induction are primarily due to changes in photochemical quenching (qP) rather than Fv′/Fm′, the ratio of variable to maximum fluorescence yield, i.e. the photochemical yield of open PSII traps in the light-adapted state [Y(II) = qP × Fv'/Fm']. They also showed that the gross rate of carbon assimilation correlated linearly with the electron transport rate through PSII determined by chlorophyll (Chl) fluorescence (ETR2fl). However, ETR2fl, obtained by fluorescence, could include any cyclic electron flux around PSII (CEF2), any charge recombination to the ground state in PSII (CR2) and any electron flux that reduces O2 during induction. These electron fluxes, though not used for carbon assimilation, may serve as photoprotective safety valves in the early induction phase. During early induction, photoprotection is most needed because PSII is highly prone to photoinactivation at that stage. Indeed, for equal cumulative light dosage (photon exposure), short pulses of actinic light (e.g. 2 s) are much more effective in photoinactivating PSII than long pulses (e.g. 100 s) during which induction is allowed to occur to some extent (Shen et al. 1996). Thus, it would be interesting to probe semiquantitatively these electron fluxes in PSII during induction. In steady-state photosynthesis, it is possible to optimize the excitation and detection of Chl fluorescence such that the fluorescence-derived electron flux through PSII (ETR2fl) can be empirically matched with 4× the gross rate of oxygen evolution in spinach, poplar and rice leaf segments up to a maximum irradiance of about 1,000 µmol·m−2·s−1 (Zhang et al. 2018). Such matching suggests that the optimized fluorescence signal represents the same tissue as in oxygen measurements at or below the maximum irradiance. However, this matching may not hold during photosynthetic induction, because various electron fluxes may exist initially before downstream carbon assimilation is fully activated. To probe the mix of electron fluxes in both photosystems during induction, we used the optimized Chl fluorescence signal, the redox signal of the primary donor of PSI (P700) and membrane inlet mass spectrometry (MIMS). The objectives of this study are to estimate time courses and magnitudes of (i) the combined CEF2 + CR2 electron flux; (ii) the rate of O2 reduction mediated by PQA (the primary quinone acceptor in PSII), PTOX (plastid terminal oxidase or plastoquinol terminal oxidase) and/or Mehler reaction; (iii) the combination of cyclic electron flux around PSI (CEF1) and any charge recombination to the ground state in PSI (CR1), with or without inhibition by antimycin A (AA) of the pathway mediated by the ‘ferredoxin-quinone reductase’ [FQR, often thought to be the Proton Gradient regulation (PGR5/PGRL1) complexes, though the exact role of PGR5/PGRL1 complexes is still being debated], or with inhibition of NDH by 2-thenoyltrifluoroacetone (TTFA); and (iv) gross O2 evolution from the water-splitting reaction and the resulting net CO2 uptake, as well as O2 uptake. To assess a possible change in the partitioning of excitation energy between both photosystems during induction, we also evaluated changes, if any, in energy partitioning by measuring P700 photo-oxidation kinetics at 77 K. The results allow partial dissection of electron fluxes in both photosystems with reasonable time resolution, which should aid in understanding photosynthetic induction and photoprotection, and in fine-tuning in silico simulations (Kroon and Thoms 2006, Deák et al. 2014, Tikhonov 2015, Antal et al. 2018) of photosynthetic electron transport events in vivo. Results Measurements of Chl fluorescence and the P700 redox signal, or of Chl fluorescence and gas exchange by MIMS were conducted on leaf disks under identical illumination conditions to ensure comparability. Testing for any change in energy partitioning between the two photosystems during photosynthetic induction Calculation of an electron flux through each photosystem using the respective photochemical yield requires, among other factors, the fraction of absorbed light partitioned to that photosystem. In steady-state photosynthesis in spinach leaf disks under broad-spectrum white light from a halogen lamp, the fraction of absorbed light partitioned to PSI (fI) is close to 0.5 (Kou et al. 2013, Fan et al. 2016). Similarly, fII in steady-state illumination with white light, obtained by a method similar to that used by Miyake et al. (2004), was close to 0.5 in spinach (as well as in poplar, rice and cotton), showing balanced excitation of the two photosystems in broad-spectrum white light (Zhang et al. 2018). However, these techniques could not be used to obtain fI or fII during photosynthetic induction if transient electron fluxes occurred in PSII without resulting in O2 exchange or P700+ reduction. In an alternative technique, we investigated whether excitation of PSI changed during induction toward steady state. We first ascertained that increasing far-red irradiance proportionally increased kox, the rate coefficient of the second-order photo-oxidation of P700 at 77 K (Satoh et al. 1976, Jia et al. 2014). Fig. 1A indicates that increased input of far-red light to PSI indeed increased kox linearly. Similarly, if the partitioning of excitation energy to PSI increased, we would expect an increase in kox. However, we observed no change in kox (measured at 77 K) during photosynthetic induction at 22°C in broad-spectrum white actinic light (Fig. 1B). Therefore, we assume the same partitioning of excitation energy during photosynthetic induction as in the steady state in spinach (0.5 to each photosystem). Fig. 1 Open in new tabDownload slide The second-order rate coefficient of P700 photo-oxidation at 77 K, as function of relative far-red irradiance (A, where the maximum irradiance was ∼64 µmol·m−2·s−1) or time of actinic illumination with broad-spectrum white light at 22°C before freezing leaf disks at 77 K (B, where the irradiance was ∼43 µmol·m−2·s−1). Values are means ± SE (n = 4–7 leaf disks). Fig. 1 Open in new tabDownload slide The second-order rate coefficient of P700 photo-oxidation at 77 K, as function of relative far-red irradiance (A, where the maximum irradiance was ∼64 µmol·m−2·s−1) or time of actinic illumination with broad-spectrum white light at 22°C before freezing leaf disks at 77 K (B, where the irradiance was ∼43 µmol·m−2·s−1). Values are means ± SE (n = 4–7 leaf disks). Electron fluxes through PSII and PSI in H2O-infiltrated leaf disks The electron flux through PSII determined by Chl fluorescence, ETR2fl, was about 35 (at irradiance 500 µmol photons·m−2·s−1, Fig. 2A, plotted semilogarithmically) and about 40 µmol electrons·m−2·s−1 (at irradiance 1,000 µmol photons·m−2·s−1, Fig. 2B) during the initial lag phase (up to induction time t = 20 s). Subsequently, ETR2fl increased steadily. At both actinic irradiances, the total electron flux through PSI, ETR1, was initially (at t = 10 s) slightly smaller than ETR2fl, but soon became larger than ETR2fl. The difference between them, ΔFlux = ETR1 − ETR2fl, includes the cyclic electron flux around PSI (CEF1) and is modulated by other electron fluxes in both photosystems. ΔFlux peaked at 40–50 s during photosynthetic induction. In steady-state illumination, ΔFlux was about 40 and 70 µmol electrons·m−2·s−1 at irradiance 500 and 1,000 µmol·m−2·s−1, respectively (Fig. 2A, B). Fig. 2 Open in new tabDownload slide Electron fluxes as a function of photosynthetic induction time. ETR1 is the electron flux through PSI, and ETR2 through PSII. ΔFlux = ETR1 − ETR2. The time axis is logarithmic. Actinic illumination of leaf disks with broad-spectrum white light at 25°C was at irradiance 500 (A) or 1,000 (B) µmol·m−2·s−1. Leaf disks had been vacuum-infiltrated with water as a control, allowed to evaporate excess water from the intercellular space in darkness and kept in darkness and a covered, humidified dish for 1–5 h before use in experiments. Values are means ± SE (n = 4–9 leaf disks). Fig. 2 Open in new tabDownload slide Electron fluxes as a function of photosynthetic induction time. ETR1 is the electron flux through PSI, and ETR2 through PSII. ΔFlux = ETR1 − ETR2. The time axis is logarithmic. Actinic illumination of leaf disks with broad-spectrum white light at 25°C was at irradiance 500 (A) or 1,000 (B) µmol·m−2·s−1. Leaf disks had been vacuum-infiltrated with water as a control, allowed to evaporate excess water from the intercellular space in darkness and kept in darkness and a covered, humidified dish for 1–5 h before use in experiments. Values are means ± SE (n = 4–9 leaf disks). Effects of AA AA is widely recognized as an inhibitor of cyclic electron transport around PSI (CEF1) mediated by a ferredoxin-dependent quinone reductase FQR (Tagawa et al. 1963, Munekage et al. 2002, Labs et al. 2016), whereas it has also been reported to inhibit CEF2 (Takagi et al. 2018). At actinic irradiance 500 µmol·m−2·s−1, AA decreased steady-state ETR1 to 70 µmol electrons·m−2·s−1 (Supplementary Fig. S1) compared with 140 µmol electrons·m−2·s−1 in the absence of AA (Fig. 2A). Similarly, at irradiance 1,000 µmol·m−2·s−1, AA decreased steady-state ETR1 to 100 µmol electrons·m−2·s−1 (Fig. 3A) compared with 220 µmol electrons·m−2·s−1 in the absence of AA (Fig. 2B). The halving of ETR1 by AA is evidence that it inhibited CEF1. Fig. 3 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 200 µM AA. Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 5 leaf disks). Fig. 3 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 200 µM AA. Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 5 leaf disks). ETR1 was close to 0 during the first 40 s when AA was present (Supplementary Fig. S1A; Fig. 3A). The slightly negative ETR1 at actinic irradiance 500 µmol·m−2·s−1 during the first 30 s in Supplementary Fig. S1A could have been due to the following factors: (i) Y(NA) was about 1 (the acceptor side of PSI was fully reduced, Supplementary Fig. S1B); (ii) Y(ND) was close to 0 (P700 was fully reduced, Supplementary Fig. S1C); and (iii) the light-induced signal was very small during the early phase of induction (3-fold smaller than the H2O control at the same induction time, data not shown). These factors mean that, in the AA treatment during the early phase of induction at irradiance 500 µmol·m−2·s−1, we were working at the limits of both the instrument and the dual-wavelength technique, such that interference from other signals became relatively prominent. Therefore, ETR1 in this figure is better treated as being only semiquantitative during the early phase of induction in the presence of AA. That ETR1 was close to 0 at this time (<40 s) suggests that forward electron transport was impaired when the Calvin–Benson cycle had not yet been substantially activated. During early induction, the fraction of PSI excitation dissipated nonphotochemically due to acceptor-side limitation, Y(NA), was markedly increased relative to the control; nevertheless, in steady state, Y(NA) decreased substantially (Supplementary Fig. S1B; Fig. 3B). The fraction of PSI excitation dissipated nonphotochemically due to donor-side limitation Y(ND) was at first much lower (near 0, i.e. P700 was almost fully reduced) in the presence of AA than in the control (Supplementary Fig. S1C; Fig. 3C). However, Y(ND) in the steady state was similar for AA treatment and for control. The blockage of downstream electron flow early during induction should result in highly reduced PQA, the primary quinone acceptor in PSII. Consistent with this expectation, the photochemical quenching parameter qP was initially very low, i.e. PQA was highly reduced (Supplementary Fig. S1D; Fig. 3D) and the photochemical yield of PSII, Y(II), was initially also low (Supplementary Fig. S1F; Fig. 3F). In contrast to ETR1 being near 0, ETR2fl was already substantial during the early phase of induction in the presence of AA (Supplementary Fig. S1A; Fig. 3A). Consequently, ΔFlux = ETR1 − ETR2fl was negative during the greater part of induction (Supplementary Fig. S1A; Fig. 3A). The fraction of PSII excitation dissipated nonphotochemically in a constitutive manner, Y(NO), was initially very large in the presence of AA (Supplementary Fig. S1G; Fig. 3G). This means that ‘constitutive’ or ‘inevitable’ dissipation (Klughammer and Schreiber 2008) of excitation energy in PSII, in a manner relatively independent of the trans-thylakoid ΔpH, was greatly enhanced. From here on, we focus on the effects of inhibitors at only a single actinic irradiance, 1,000 µmol·m−2·s−1. Effects of TTFA, an inhibitor of NDH, in the absence or presence of AA TTFA (2-thenoyltrifluoroacetone) inhibits NDH and NDH-dependent CEF1 (Mi et al. 1994). By itself, TTFA had little or no effect on ETR2fl (Fig. 4A; compare with the control in Fig. 2B), nor on other PSII parameters (Fig. 4D–G), when compared with H2O treatment. It did, however, slightly decreased Y(NA) (Fig. 4B) and increased Y(ND) (Fig. 4C). In the presence of TTFA alone, ETR1 > ETR2fl (Fig. 4A), so that ΔFlux was positive at all times during the 10-min illumination. Fig. 4 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 100 µM 2-thenoyltrifluoroacetone (TTFA). Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 3 leaf disks). Fig. 4 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 100 µM 2-thenoyltrifluoroacetone (TTFA). Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 3 leaf disks). In the presence of a combination of AA and TTFA, both the FQR- and NDH-dependent pathways of CEF1 (Johnson 2011) would have been inhibited, and both ETR2 and ETR1 were markedly decreased (Fig. 5A) compared with the control (Fig. 2B). ΔFlux was near 0 for the first 2 min (Fig. 5A). Fig. 5 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 200 µM AA + 100 µM TTFA. Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 4 leaf disks). Fig. 5 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 200 µM AA + 100 µM TTFA. Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 4 leaf disks). Y(NA) in the presence of AA + TTFA was high during the first 2–3 min of induction (Fig. 5B). Nevertheless, Y(NA) in the presence of AA + TTFA (Fig. 5B) was not as high as in the presence of AA alone (Fig. 3B). Y(ND) in the presence of AA + TTFA was very low, relative to the H2O treatment (Fig. 5C). Nevertheless, Y(ND) (Fig. 5C) was slightly greater than in the presence of AA alone (Fig. 3C). In terms of PSII, Y(II) in the presence of AA + TTFA was lower than that of the H2O control (Fig. 5F), accompanied by very low qP (Fig. 5D), as expected from Y(II) = qP × Fv′/Fm′. Notably, Fv′/Fm′ was slightly greater in the combined presence of AA + TTFA than in the control (Fig. 5E). Effects of methyl viologen CEF1 can also be eliminated by methyl viologen (MV) which rapidly mediates electron flow from PSI to O2. Further, in the presence of MV, little or no charge recombination to the ground state in PSI (CR1) is expected, because electrons are rapidly cleared from the acceptor side. Although oxygen reduction is facilitated by MV, ETR1 was very low during induction (Fig. 6A); indeed, ETR1 was smaller than ETR2fl which included any PSII electron fluxes that bypassed PSI. As these PSII electron fluxes declined during photosynthetic induction, ΔFlux approached 0; i.e. ETR1 ≈ ETR2fl in steady-state illumination in the presence of MV. Fig. 6 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 100 µM MV. Actinic illumination was at irradiance 1,000 µmol·m−2·−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 3 leaf disks). Fig. 6 Open in new tabDownload slide (A) Electron fluxes as a function of photosynthetic induction time. Leaf disks had been vacuum-infiltrated with 100 µM MV. Actinic illumination was at irradiance 1,000 µmol·m−2·−1. Other conditions are the same as in Fig. 2. PSI parameters (B, C) and PSII parameters (D–G) at various induction times were measured. Values are means ± SE (n = 3 leaf disks). As electrons are cleared rapidly from the acceptor side of PSI, as expected in the presence of MV, Y(NA) was very small throughout the induction (Fig. 6B). At the same time, Y(ND) was very high (Fig. 6C) as P700 was readily photo-oxidized in the presence of MV, whereas electron supply from upstream was slow. Perhaps surprisingly, qP was low throughout induction (Fig. 6D); i.e. PQA was in a highly reduced state. Concomitantly, Y(II) was also very low during induction (Fig. 6F). It is also significant that in the presence of MV, Y(NPQ) was high right from the start of illumination and remained high throughout (Fig. 7). Fig. 7 Open in new tabDownload slide The energy-dependent NPQ parameter Y as a function of induction time for leaf disks preinfiltrated with H2O, 100 µM MV or 200 µM AA. Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. Values are means ± SE (n = 3–5 leaf disks). Fig. 7 Open in new tabDownload slide The energy-dependent NPQ parameter Y as a function of induction time for leaf disks preinfiltrated with H2O, 100 µM MV or 200 µM AA. Actinic illumination was at irradiance 1,000 µmol·m−2·s−1. Other conditions are the same as in Fig. 2. Values are means ± SE (n = 3–5 leaf disks). MIMS measurements To further elucidate the electron fluxes during photosynthetic induction, we measured the O2 and CO2 fluxes by MIMS. There was only a little gross oxygen evolution from water splitting for the first 30 s (Fig. 8A, in which both axes are logarithmic). Both gross oxygen evolution (Fig. 8A) and net CO2 uptake (Fig. 8C) accelerated after about 2 min. As expected, 3-3,4(dichlorophenyl)-1,1-dimethyl-urea (DCMU) almost completely abolished both gross oxygen evolution (Fig. 8A) and carbon assimilation (Fig. 8C). Fig. 8 Open in new tabDownload slide (A) The gross rate of oxygen evolution from water-splitting as a function of induction time. Both axes are logarithmic. (B) The net O2 uptake rate, after the mitochondrial dark respiration rate has been subtracted, plotted as a function of induction time. The time axis is logarithmic. (C) The net CO2 uptake rate, with mitochondrial respiration included, plotted as a function of induction time (logarithmic scale). In (B, C), the y-axis is linear. Values are means ± SE (n = 6–9 leaf disks). Fig. 8 Open in new tabDownload slide (A) The gross rate of oxygen evolution from water-splitting as a function of induction time. Both axes are logarithmic. (B) The net O2 uptake rate, after the mitochondrial dark respiration rate has been subtracted, plotted as a function of induction time. The time axis is logarithmic. (C) The net CO2 uptake rate, with mitochondrial respiration included, plotted as a function of induction time (logarithmic scale). In (B, C), the y-axis is linear. Values are means ± SE (n = 6–9 leaf disks). The rate of chloroplast O2 uptake, calculated as the gross oxygen uptake rate minus the dark (mitochondrial) respiration rate, represents chloroplast O2 reduction mediated by the Mehler reaction, PTOX reaction and/or PQA. The rate of chloroplast O2 reduction was near 0 at induction time t = 10 s (Fig. 8B). The peak rate of chloroplast O2 uptake at about 40 s was similar in H2O- and AA-treatments, being approximately 5 µmol O2·m−2·s−1, equivalent to approximately 10 µmol electrons·m−2·s−1 (Fig. 8B). The peak was considerably lowered by n-propyl gallate (PG) which inhibits PTOX (Cournac et al. 2000, Stepien and Johnson 2009). The peak rate of chloroplast O2 reduction was also lowered by DCMU, to a similar extent as by n-PG (Fig. 8B). To further analyze the electron fluxes in and around PSII, the sum of the cyclic electron flux around PSII and charge recombination to the ground state (CEF2 + CR2) was calculated as the difference between ETR2fl and 4× gross O2 evolution rate (Fig. 9). At t = 40 s, CEF2 + CR2 was between 30 and 40 µmol electrons·m−2·s−1. At longer induction times, there was a trend of CEF2 + CR2 toward 0 at steady state. Comparing CEF2 + CR2 in H2O control and AA treatment, there was no difference within the first minute (Fig. 9). Fig. 9 Open in new tabDownload slide Estimation of CEF2 + CR2, obtained as the difference ETR2fl − 4× gross O2 evolution rate, plotted as a function of induction time. Fig. 9 Open in new tabDownload slide Estimation of CEF2 + CR2, obtained as the difference ETR2fl − 4× gross O2 evolution rate, plotted as a function of induction time. Discussion Constancy of energy partitioning between the two photosystems during photosynthetic induction Conventional methods used in steady-state photosynthesis to obtain fI and fII could not be used during photosynthetic induction. However, we observed that the second-order rate coefficient for P700 photo-oxidation measured at 77 K (kox) was constant during photosynthetic induction at 22°C, implying that excitation of PSI was unchanging. The constancy of kox during induction is consistent with the report of Wood et al. (2018) that upon transition from darkness to white growth light (200 µmol·m−2·s−1), there was no significant change in the size of the PSI or PSII antenna as judged by the excitation spectra at 77 K, and only a minor change in the PSI emission at 735 nm was observed. Further, they found that transition from darkness to growth light did not trigger any redistribution of LHCII between grana and stromal lamellae, unlike in state transition brought about by colored lights. Similarly, earlier work (Tikkanen et al. 2010, Mekala et al. 2015) showed that changes in PSII and LHCII phosphorylation induced by varying the irradiance of white light do not significantly change the antenna size of either photosystem in plants. Thus, for calculating ETR1 and ETR2fl, we assumed that fI = fII = 0.50 (a value previously measured in spinach during steady-state photosynthesis) throughout photosynthetic induction. Electron fluxes through PSII and PSI in H2O-infiltrated leaf disks The difference between the electron flux through PSI and that through PSII, ΔFlux = ETR1 − ETR2fl, includes any cyclic electron flux around PSI (CEF1) and is modulated by other electron fluxes in both photosystems. It peaked at 40–50 s during photosynthetic induction; this peak could be mainly due to a peak in CEF1. The previous estimation of CEF1 by Fan et al. (2007), using red + far-red actinic light (680–750 nm) and the initial rate of postillumination P700+ re-reduction, also showed a peak, at induction time t ≈ 30 s. Simulation of induction events produced a peak in CEF1 at t ≈ 2 s (Tikhonov 2015), an order of magnitude earlier. In another study, Vredenberg and Bulychev (2010) simulated the redox induction kinetics of P700 and of CEF1; CEF1 reached a maximum at t = 10 s, the longest far-red illumination time used. Obviously, experimental conditions need to be taken into account, in addition to the adjustment of simulation parameters, to enable matching of simulated and experimental induction kinetics of CEF1. After a dip at about t ≈ 80 s, ΔFlux at each actinic irradiance increased for about a minute before plateauing (Fig. 2). By contrast, simulations indicate that CEF1 peaks and then declines to a steady value monotonically, without any dip (Tikhonov 2015). However, we cannot simply equate ΔFlux with CEF1 during photosynthetic induction: ΔFlux could be contaminated by other electron fluxes, such as a transient component of ETR2fl that is subtracted from ETR1 to give ΔFlux. To delve into the components that made up ΔFlux, we used a variety of inhibitors and/or mediators of electron transport or oxygen reduction. Effects of AA At both 500 and 1,000 µmol photons·m−2·s−1, ETR2fl was positive during induction in the presence of AA, even though ETR1 was near 0 initially (Supplementary Fig. S1A; Fig. 3A); this resulted in ΔFlux being negative during the greater portion of the induction. This interesting observation suggests that other electron fluxes occurred around/near PSII but that they were shunted away from reaching PSI (ETR1 being near 0 initially). We term them collectively a shunted electron flux (SEF), estimated as the magnitude of the difference ΔFlux = ETR1 − ETR2fl if CEF1 were completely inhibited by AA, as seems to be the case in steady-state illumination when ΔFlux was near 0 (Supplementary Fig. S1A; Fig. 3A). Notably, ΔFlux was negative for the first several minutes during induction; this suggests that SEF was transient, declining as the Calvin–Benson cycle became activated. SEF could have resulted from (i) electrons accumulating upstream of PSI, thereby enhancing a local cyclic flux around PSII (CEF2), (ii) charge recombination in PSII to the ground state (CR2) and/or (iii) electron transfer to oxygen before PSI, via plastoquinonol terminal oxidase (PTOX; Tikhonov 2015) or via PQA (Cleland and Grace 1999). The fraction of PSII excitation dissipated nonphotochemically in a constitutive manner Y(NO) was initially very large in the presence of AA (Supplementary Fig. S1G; Fig. 3G). This ‘constitutive’ or ‘inevitable’ dissipation (Klughammer and Schreiber 2008) of excitation energy in PSII, in a manner relatively independent of the trans-thylakoid ΔpH, could come about in at least three ways. First, an internal cyclic electron flux within PSII, such as CR2 (charge recombination to the ground state) and/or CEF2 which delivers an electron from the secondary quinone acceptor PQB via cytochrome b559 to the donor side of PSII (Takagi et al. 2018), could compete against photochemical conversion without contributing to ΔpH across the thylakoid membrane. Indeed, ΔpH [as indicated by Y(NPQ)] was negligibly small during the first 30 s of photosynthetic induction in the presence of AA (Fig. 7). As it has been reported that AA inhibits CEF2 (Takagi et al. 2018), it is likely that CR2, rather than CEF2, was responsible for the initially high Y(NO) in Supplementary Fig. S1G and Fig. 3G. Second, there could be electron transfer from PSII to oxygen via PTOX (Peltier et al. 2010, Stepien and Johnson 2018) or PQA (Cleland and Grace 1999) that elevated dissipative Y(NO) without contributing to ΔpH. Given that PTOX is located on the stromal face of unstacked regions of the thylakoid membrane (Lennon et al. 2003), PTOX-mediated reduction of O2 by PQH2 should not enhance ΔpH. Third, dissipative Y(NO) could come from frequent charge recombination to the excited state in PSII followed by charge separation according to the exciton-radical pair equilibrium model (Trissl and Lavergne 1995): each charge separation followed by charge recombination may incur a slight loss of photochemical conversion efficiency, and multiple repeats before the charge is stabilized leads to a substantial Y(NO); this was an explanation proposed for Y(NO) (Hendrickson et al. 2004). A downside of this third mechanism is that electron spin reversal may lead to triplet excited states of chlorophyll that interact with O2, thereby generating singlet oxygen that is highly damaging to PSII (Vass 2011). Indeed, this mechanism well explains why long delays in the arrival of a second electron at PQB (a two-electron gate), due to either low continuous irradiance or widely spaced single-turnover flashes, promotes recombination of PQB− with the S2,3 states of the oxygen-evolving complex. The long-waiting period before the arrival of a second electron at PQB− presumably increases the chance of electron spin reversal and formation of triplet excited states upon recombination of PQB− with the S2,3 states, thereby greatly increasing the quantum efficiency of PSII photoinactivation (Keren et al. 1995, Keren et al. 1997). Y(II) was decreased by AA treatment (Supplementary Fig. S1F; Fig. 3F), relative to the H2O control, by a percentage much smaller than that by which qP was decreased (Supplementary Fig. S1D; Fig. 3D), probably because Fv′/Fm′ was increased (Supplementary Fig. S1E; Fig. 3E) relative to the control, thereby compensating for the decrease in qP in the calculation of Y(II) = qP × Fv′/Fm′. An increase in Fv′/Fm′ means that the photochemical conversion efficiency of open PSII traps increases; this could come about if some excitation energy from closed PSII traps were transferred to open PSII traps that are excitonically connected. In PSI, the fraction of PSI excitation dissipated nonphotochemically due to acceptor-side limitation Y(NA) was markedly increased relative to the control (Supplementary Fig. S1B; Fig. 3B). This can be explained by AA inhibiting CEF1, such that electrons could not be diverted from the acceptor side of PSI back to the PQ pool via FQR. Nevertheless, under such inhibitory conditions, charge recombination to the ground state in PSI (CR1) could occur, thereby keeping P700 highly reduced initially (Supplementary Fig. S1C; Fig. 3C). In turn, this had a feedback effect on intersystem electron carriers, so that the PQ pool, PQB and PQA were more reduced initially (qP of AA treatment < qP of control, Supplementary Fig. S1D; Fig. 3D). In steady state, however, Y(NA) decreased substantially and qP increased markedly. It appears that, even in the presence of AA, electrons were flowing through PS-I more freely in steady-state illumination, as the Calvin–Benson cycle was now activated. Indeed, Y(II) (and, therefore, ETR2fl) increased by a factor of about three during photosynthetic induction at each irradiance (Supplementary Fig. S1F; Fig. 3F). The fraction of PSI excitation dissipated nonphotochemically due to donor-side limitation Y(ND) was at first much lower (near 0) in the presence of AA than in the control (Supplementary Fig. S1C; Fig. 3C). It appears that inhibition of CEF1 resulted in markedly increased Y(NA) and hence increased charge recombination within PSI to keep P700 highly reduced during early induction. However, Y(ND) in the steady state was similar for AA treatment and for control. This may be associated with the free linear flow of electrons in steady state, as the Calvin–Benson cycle became activated. With the decline in Y(NA) as induction progressed, despite the increase in Y(ND), charge recombination to the ground state in PSI (CR1) appeared to decline, because ΔFlux was close to 0 in steady-state illumination (Supplementary Fig. S1A; Fig. 3A). Effects of TTFA, an inhibitor of NDH, in the absence or presence of AA By itself, TTFA had little or no effect on PSII parameters (Fig. 4A, D–G), though it slightly increased Y(ND) (Fig. 4C) and decreased Y(NA) (Fig. 4B) compared with the H2O control. Indeed, Y(NA) was, within scatter, close to 0 during much of the photosynthetic induction phase, reflecting a highly oxidized acceptor side of PSI. By contrast, in the presence of a combination of AA and TTFA, when both the FQR- and NDH-dependent pathways of CEF1 (Johnson 2011) would have been inhibited, both ETR2 and ETR1 were markedly decreased (Fig. 5A) compared with the control (Fig. 2B). ΔFlux was near 0 for the first 2 min in the combined presence of both inhibitors, compared with about −30 µmol·m−2·s−1 in the presence of AA alone (Fig. 3A). That ΔFlux was not negative in the combined presence of TTFA and AA was probably due to increased charge recombination in PSI in the combined presence of TTFA + AA (tending to elevate ETR1 and ΔFlux) compared with AA treatment alone, thereby compensating for the SEF (included in ETR2fl) which tended to lower the magnitude of ΔFlux when ETR1 > ETR2fl. As SEF gradually declined during activation of the Calvin–Benson cycle beyond 2 min (albeit in a limited fashion, see Fig. 8C), ΔFlux increased, probably because enhanced charge recombination within PSI, in the combined presence of AA and TTFA, contributed to ETR1, making ΔFlux more and more positive (Fig. 5A). The observation that TTFA had little or no effect on PSII parameters (Fig. 4A, D–G), though it slightly increased Y(ND) (Fig. 4C) and decreased Y(NA) (Fig. 4B), suggests that it could have a regulatory role in electron transport in spinach leaves. Here, we propose the following working hypothesis that NDH assists the FQR-dependent, AA-sensitive CEF1, specifically by facilitating forward electron transfer from the Cyt bf complex toward PSI. In accordance with this hypothesis, when NDH was inhibited by TTFA, the AA-sensitive electron flux (ΔFluxTTFA − ΔFluxTTFA+AA) was much smaller than the AA-sensitive electron flux (ΔFluxH2O − ΔFluxH2O+AA) of the control (compare Fig. 10A with Fig 10B), at a given actinic irradiance. Further, when NDH was inhibited by TTFA, the FQR-dependent, AA-sensitive CEF1 occurred only briefly during photosynthetic induction, and disappeared within 5 min at irradiance 1,000 µmol·m−2·s−1 (ΔFluxTTFA − ΔFluxTTFA+AA approaching 0) (Fig. 10A); at irradiance 500 µmol·m−2·s−1, it took longer for ΔFluxTTFA − ΔFluxTTFA+AA to approach 0 than at the higher irradiance (Fig. 10A). Perhaps the redox poise for FQR-dependent, AA-sensitive CEF1 at the higher irradiance was more quickly disrupted upon inhibition of NDH. Indeed, Martin et al. (2015) proposed that the NDH complex improves CEF1 by adjusting the redox level of electron transporters. Fig. 10 Open in new tabDownload slide The AA-sensitive component of ΔFlux, calculated as the difference between ΔFlux in the presence and that in the absence of AA in a background treatment with TTFA (A) or H2O (B), plotted against induction time under illumination at 500 (closed circles) or 1,000 (open triangles) µmol·m−2·s−1. Fig. 10 Open in new tabDownload slide The AA-sensitive component of ΔFlux, calculated as the difference between ΔFlux in the presence and that in the absence of AA in a background treatment with TTFA (A) or H2O (B), plotted against induction time under illumination at 500 (closed circles) or 1,000 (open triangles) µmol·m−2·s−1. A corollary of the present working hypothesis is that inhibition of NDH by TTFA would slow forward electron transfer from the Cyt bf complex toward P700, which consequently became more oxidized [i.e. Y(ND) was increased relative to the H2O control, Fig. 4C]. The slowing of electron flow from the Cyt bf complex to P700 could have been responsible for Y(NA) being smaller than that in the H2O control (Fig. 4B). Similarly, the combined presence of TTFA and AA decreased Y(NA) slightly (Fig. 5B) and increased Y(ND) slightly (Fig. 5C) compared with AA alone (Fig. 3B, C). This hypothesis could work if NDH, in addition to transferring an electron to PQ, could also transfer an electron to a semiplastoquinol PQi•− bound to the high potential side of the Cyt bf complex near the stroma (see Fig. 11). As PQi, like PQB, is a two-electron gate, rapid acquisition of the second electron (from NDH) would speed up the Q cycle, thereby also speeding up the reduction of Cyt f. This is likely to be more important at low actinic irradiance when the arrival of electrons to PQi is less frequent. Indeed, the AA-sensitive component of CEF1 in Arabidopsis leaf disks at (near growth irradiance) 130 µmol photons·m−2·s−1 was approximately 18 µmol electrons·m−2·s−1 for the wild type, but only about approximately 3 µmol electrons·m−2·s−1 for the ndh mutant (Kou et al. 2015). NDH is a large complex that may have various properties. For example, Strand et al. (2017) reported that it functions as a high-efficiency proton pump that increases ATP production by cyclic electron flow. Fig. 11 Open in new tabDownload slide A scheme of pathways of electron fluxes (J) around PSII, the cytochrome bf complex and PSI. It shows (i) water splitting at the water-oxidizing complex (WOC), charge recombination (CR2), cyclic electron flux (CEF2), oxygen reduction via PQA and via PTOX in/near PSII; (ii) the ‘Q cycle’ and forward electron transfer in the Cyt bf complex to plastocyanin Pc; (iii) linear electron transfer from PSI to NADP+ to drive the Calvin–Benson cycle (CBC) or to reduce O2 as part of the water–water cycle (WWC); and (iv) cyclic electron flow mediated by FQR and NDH [redrawn from Tikhonov (2015), in which other abbreviations can be found]. For simplicity, the names of some components have been left out. Fig. 11 Open in new tabDownload slide A scheme of pathways of electron fluxes (J) around PSII, the cytochrome bf complex and PSI. It shows (i) water splitting at the water-oxidizing complex (WOC), charge recombination (CR2), cyclic electron flux (CEF2), oxygen reduction via PQA and via PTOX in/near PSII; (ii) the ‘Q cycle’ and forward electron transfer in the Cyt bf complex to plastocyanin Pc; (iii) linear electron transfer from PSI to NADP+ to drive the Calvin–Benson cycle (CBC) or to reduce O2 as part of the water–water cycle (WWC); and (iv) cyclic electron flow mediated by FQR and NDH [redrawn from Tikhonov (2015), in which other abbreviations can be found]. For simplicity, the names of some components have been left out. Effects of MV ETR1 was only 30–35 µmol electrons·m−2·s−1 during photosynthetic induction (Fig. 6A). Why was ETR1 so small despite the presence of MV? As MV outcompeted against NADP+ for electrons, there would have been a shortage of NADPH for carbon assimilation and, therefore, a surplus of unconsumed ATP, accompanied by a shutdown of ATP synthesis. Consequently, the pH gradient would become very large [consistent with a very high Y(NPQ) right from the start of measurement in the presence of MV (Fig. 7)], resulting in slow PQH2 oxidation and accumulation of electrons at PQB and PQA [consistent with a persistently low qP (Fig. 6D) and Y(II) (Fig. 6F)]. This effect then slows coupled linear electron transport in leaf disks, a situation different from that of isolated lettuce thylakoids in which the uncoupled rate of electron transport mediated by MV is increased by as much as 7-fold compared with the absence of an uncoupler (Chow 1984). Apart from the back pressure of a highly acidic lumen against the oxidation of PQH2, a highly reduced PQ pool would also impair the Q cycle in the cytochrome bf complex, with concomitant impairment of linear electron transfer to Cyt f and plastocyanin Pc. This is termed reduction-induced suppression of electron flow (RISE) (Shaku et al. 2015, Shimakawa et al. 2018). Given the restriction of electron delivery to PSI, both the acceptor side (Fig. 6B) and donor side (Fig. 6C) were highly oxidized. In this study, MV had qualitatively similar effects on PSII as reported by Fan et al. (2009). However, qP in the present study was much lower, possibly because of the high actinic irradiance (broad-spectrum halogen light at 1,000 µmol·m−2·s−1) used. In the study of Fan et al. (2009), the actinic light in the 650–740 nm wavelength range, with a peak at 697 nm and an irradiance of 500 µmol·m−2·s−1, excited PSI more than PSII, so that qP was not as low as in the present study. There was a possibility that prolonged illumination at high irradiance in the presence of MV produced excessive quantities of ROS; the ROS could then overwhelm the ROS-scavenging systems. However, this possibility was ruled out, because Pm was not decreased after 15-min induction (data not shown). Thus, there was no sign of damage to PSI, for damage to PSI would have decreased Pm. Apparently, PSI was photoprotected by the RISE-restricted delivery of electrons to it. Further, a highly oxidized P700 pool [Y(ND) ≥ 0.9] would act as an efficient quencher of excitation (Shubin et al. 2008) to limit electron transport through PSI to the acceptor side in the first place. MIMS measurements The gross rate of oxygen evolution (Fig. 8A, logarithmic y-axis) and the rate of net CO2 uptake (Fig. 8C) did not accelerate until after about 2 min; during this lag phase, photoprotection is most needed. A combination of AA and TTFA drastically diminished both steady-state gross oxygen evolution (Fig. 8A) and steady-state net carbon assimilation (Fig. 8C). This is probably a result of diminished electron flow beyond PSI, because PQA was highly reduced (low qP, Fig. 5D) and ETR2fl was very low (Fig. 5A), and the acceptor side of PSI was also highly reduced [high Y(NA), Fig. 5B]. The difference in chloroplast O2 uptake rate in the absence and presence of n-PG represents the PTOX-dependent component, which peaked at about 40 s (Fig. 8B). It has been reported that plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport, though the mechanism of translocation remains to be elucidated (Stepien and Johnson 2018). The occurrence of the peak of the difference signal at about 40 s should allow sufficient time for such protein translocation, given that, e.g. segregation of PSII from PSI during Mg2+-induced thylakoid stacking at room temperature was substantial in 6 s after adding MgCl2 (Rubin et al. 1981). The peak rate of chloroplast O2 reduction was also lowered by DCMU, to a similar extent as by n-PG (Fig. 8B). This observation suggests that the residual in vivo O2 reduction was PQA-mediated, just as Cleland and Grace (1999) also reported for isolated thylakoids. The sum of the cyclic electron flux around PSII and charge recombination to the ground state (CEF2 + CR2) was calculated as the difference between ETR2fl and 4× gross O2 evolution rate (Fig. 9). At t = 40 s, CEF2 + CR2 was between 30 and 40 µmol electrons·m−2·s−1. The gross O2 evolution rate at t = 40 s was equivalent to only about 5 µmol electrons·m−2·s−1 (∼1.3 µmol O2·m−2·s−1, Fig. 8A), much smaller than CEF2 + CR2. Therefore, it is likely that CEF2 + CR2 was not oxygenic, but merely serving as a safety valve. Comparing CEF2 + CR2 in H2O control and AA treatment, there was no difference within the first minute (Fig. 9); any inhibition of CEF2 by AA (Takagi et al. 2018) could have been offset by an equivalent increase in CR2 as a photoprotective mechanism. Perhaps this concomitant increase in CR2 boosted ETR2 to such an extent that ΔFlux remained negative before the completion of induction (Supplementary Fig. S1A; Fig. 3A). At longer induction times, there was a trend of CEF2 + CR2 toward 0 at steady state; it appears that CEF2 + CR2 declined gradually during induction, as downstream processes became activated. Unfortunately, the exact time course cannot be ascertained, given that the measurements of ETR2fl and gross O2 evolution are complicated by very different time resolutions. How does CEF1 vary during photosynthetic induction? ΔFluxH2O is the net result of CEF1 and the SEF that bypasses PSI during induction. ΔFluxAA includes no CEF1 but has the SEF. The SEF seemed to be similar in magnitude in the presence and absence of AA, because (i) the rate of chloroplast O2 reduction was similar (Fig. 8B) and (ii) CEF2 + CR2 was similar (Fig. 9), at least for the first minute. Therefore, the difference ΔFluxH2O − ΔFluxAA represents the AA-sensitive CEF1 in a control leaf disk. Fig. 10B shows an initial increase in ΔFluxH2O − ΔFluxAA that peaked at approximately 40 s. During this period, carbon assimilation had not yet been substantially activated, and CEF1 was probably playing a photoprotective role (Takahashi et al. 2009, Alboresi et al. 2019). From t ≈ 120–160 s onward (depending on actinic irradiance), as linear electron transport began to accelerate (evident from gas-exchange data, Fig. 8A, C), CEF1 began to decline (Fig. 10B). This confirms the findings of Wood et al. (2018) who concluded that transition from darkness to growth light-induced structural changes [resulting in more but smaller grana, also reported by Rozak et al. (2002) and Anderson et al. (2012)], which enhanced linear electron transport, whereas CEF1 is favored by the opposite structural changes. In summary, this study has demonstrated partial dissection of the electron fluxes in and around both photosystems in spinach leaf disks during photosynthetic induction. In particular, the peak magnitudes of some of the electron fluxes depicted in Fig. 11, at 1,000 µmol photons·m−2·s−1, are in the descending order: CEF1 (∼110 µmol electrons·m−2·s−1) > CEF2 + CR2 (∼40 µmol electrons·m−2·s−1) > chloroplast O2 uptake (∼10 µmol electrons·m−2·s−1). These electron fluxes most probably served a photoprotective role when the Calvin–Benson cycle was not yet substantially activated. The magnitudes and time courses of these electron fluxes will help to fine-tune in silico simulation models of electron transport processes in vivo. Materials and Methods Plant growth and vacuum infiltration of leaf disks Spinacia oleracea L. (cv. Yates hybrid 102) plants were grown in a greenhouse as described previously (Kou et al. 2013). Leaf disks (1.97 cm2) were vacuum-infiltrated with H2O, AA (200 µM), 2-thenoyltrifluoroacetone (TTFA, 100 µM), n-PG (1 mM), MV (100 µM) or DCMU (100 µM). Excess intercellular water in leaf disks was allowed to evaporate in darkness until they were no longer translucent, and then the leaf disks were kept on moist filter paper in a covered Petri dish. The total dark time was approximately between 1 and 5 h before leaf disks were illuminated at either 500 or 1,000 µmol photons·m−2·s−1 as specified in the text. Measurement of the electron flux through PSII by Chl fluorescence (ETR2fl) Chl fluorescence yield was measured with a PAM 101–103 fluorometer (Walz) as described by Zhang et al. (2018). A leaf disk was placed in an oxygen electrode chamber at 25°C in air enriched with 1% CO2. The modulated green excitation irradiance was 0.34 µmol·m−2·s−1. At 10-s intervals, a saturating pulse (0.8-s duration, 7,000 µmol·m−2·s−1) was applied to determine the maximum fluorescence yield in the light-adapted state (Fm′). The photochemical yield of PSII, Y(II), was determined as 1 − F/Fm′, where F is the fluorescence yield at a given induction time. ETR2fl was obtained as Y(II) × I × 0.85 × fII, where I is the irradiance, 0.85 is the assumed leaf absorptance and fII is the fraction of the absorbed light partitioned to PSII. The photochemical quenching parameter qP was obtained as described by Zhang et al. (2018), the energy-dependent non-photochemical quenching of excitation Y(NPQ) was determined according to Hendrickson et al. (2004) and the constitutive dissipation of excitation in PSII was obtained as Y(NO) = 1 − Y(II) − Y(NPQ). The photochemical yield of open PSII traps in the light-adapted state (Fv′/Fm′) was obtained as Y(II)/qP. Measurement of the electron flux through PSI by the redox kinetics of P700 (ETR1) The photochemical yield of PSI, Y(I), was obtained using a dual-wavelength unit (ED-P700DW, Walz) as described by Zhang et al. (2018). The extent (P) of oxidation of P700 during actinic illumination (500 or 1,000 µmol·m−2·s−1, as specified), as well as the maximum oxidation of P700 (Pm′) upon superimposing a strong far-red pulse plus a saturating white pulse, was recorded every 10 s during early induction and less frequently later. The maximum extent of P700 oxidation (Pm) in the presence of continuous weak far-red light was determined by adding a saturating pulse (0.5-ms duration, 10,000 µmol photons·m−2·s−1 from a white light-emitting diode); this was performed within a few minutes after actinic illumination. Y(I) was calculated as (Pm′ − P)/Pm (Klughammer and Schreiber 2007). ETR1 was obtained as Y(I) × I × 0.85 × fI, where fI (= 1 − fII) is the fraction of the absorbed light partitioned to PSI. NPQ of excitation in PSI due to donor-side limitation in PSI was obtained as Y(ND) = P/Pm, and acceptor-side limitation obtained as Y(NA) = 1− Y(I) − Y(ND). Measurement of the second-order rate coefficient of P700 photo-oxidation at 77 K The second-order rate coefficient of P700 photo-oxidation (kox), a measure of the partitioning of energy to PSI, was determined at 77 K where electron donation to P700+ was absent, using the method of Jia et al. (2014). Leaf disks (1.65 cm2) were illuminated for a defined duration of induction at 22°C in air containing 1% CO2, and then frozen at 77 K; kox was given by the slope of a plot of 1/[P700reduced] against time of illumination with weak far-red light (725 nm, 43 µmol·m−2·s−1). Membrane inlet mass spectrometry Gas exchange was measured with a purpose-built cuvette connected to a membrane inlet mass spectrometer (Micromass ISOPRIME, Micromass Ltd., Manchester, UK) which was operated in peak switching mode for 18O2, 16O2 and CO2, as described by Maxwell et al. (1998). A leaf disk (1.3 cm2) was placed in the closed cuvette at 25°C. After calibrating for oxygen, the cuvette was flushed with nitrogen, and 20 µl of CO2 was injected to calibrate the CO2 signal, giving about 4% CO2 in an atmosphere containing 18–21% O2. Net carbon uptake was measured from the decline in CO2 concentration. Gross O2 evolution and gross O2 uptake were calculated from changes in 16O2 and 18O2, respectively. A thermal effect from the white halogen light, obtained with a green card disk that resembled a leaf disk in thickness and color, was subtracted from the rates of gas exchange. A multifurcated light guide enabled Chl fluorescence yield to be monitored concurrently. Acknowledgments We thank Assoc. Prof. Oula Ghannoum for the gift of TTFA and Prof. Owen Atkin for the PG used in the inhibitor experiments. Funding M.Z. gratefully acknowledges the award of a scholarship from the China Scholarship Council that supported her research. This research was also supported by the Australian Government through the Australian Research Council Centre of Excellence for Translational Photosynthesis [CE1401000015 to M.R.B.]; the National Key Research and Development Program of China [2016YFA0600802 to D.-Y. F.]; the National Natural Science Foundation of China [31870373 to G.-Y.S.]. Disclosures The authors have no conflicts of interest to declare. References Alboresi A. , Storti M. , Morosinotto T. ( 2019 ) Balancing protection and efficiency in the regulation of photosynthetic electron transport across plant evolution . New Phytol. 221 : 105 – 109 . Google Scholar Crossref Search ADS PubMed WorldCat Allee W.C. ( 1926 ) Measurement of environmental factors in the tropical rain forest of Panama . Ecology 7 : 273 – 302 . Google Scholar Crossref Search ADS WorldCat Anderson J.M. , Horton P. , Kim E.-H. , Chow W.S. ( 2012 ) Towards elucidation of dynamic structural changes of plant thylakoid architecture . Phil. Trans. R. Soc. B 367 : 3515 – 3524 . Google Scholar Crossref Search ADS WorldCat Antal T.K. , Maslakov A. , Yakovleva O.V. , Krendeleva T.E. , Riznichenko G.Y. , Rubin A.B. ( 2018 ) Simulation of chlorophyll fluorescence rise and decay kinetics, and P700-related absorbance changes by using a rule-based kinetic Monte-Carlo method . Photosynth. Res. 138 : 191 – 206 . Google Scholar Crossref Search ADS PubMed WorldCat Chow W.S. ( 1984 ) The extent to which the spatial separation of Photosystems I and II associated with granal formation limits noncyclic electron flow in isolated lettuce chloroplasts . Arch. Biochem. Biophys . 232 : 162 – 171 . Google Scholar Crossref Search ADS PubMed WorldCat Cleland R.E. , Grace S.C. ( 1999 ) Voltammetric detection of superoxide production by photosystem II . FEBS Lett. 457 : 348 – 352 . Google Scholar Crossref Search ADS PubMed WorldCat Cournac L. , Redding K. , Ravenel J. , Rumeau D. , Josse E.-M. , Kuntz M. ( 2000 ) Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration . J. Biol. Chem. 275 : 17256 – 17262 . Google Scholar Crossref Search ADS PubMed WorldCat Deák Z. , Sass L. , Kiss É. , Vass I. ( 2014 ) Characterization of wave phenomena in the relaxation of flash-induced chlorophyll fluorescence yield in cyanobacteria . Biochim. Biophys. Acta 1837 : 1522 – 1532 . Google Scholar Crossref Search ADS PubMed WorldCat Deans R.M. , Brodribb T.J. , Busch F.A. , Farquhar G.D. ( 2019 ) Plant water-use strategy mediates stomatal effects on the light induction of photosynthesis . New Phytol. 222 : 382 . Google Scholar Crossref Search ADS PubMed WorldCat Fan D.-Y. , Fitzpatrick D. , Oguchi R. , Ma W. , Kou J. , Chow W.S. ( 2016 ) Obstacles in the quantification of the cyclic electron flux around Photosystem I in leaves of C3 plants . Photosyn. Res. 129 : 239 – 251 . Google Scholar Crossref Search ADS PubMed WorldCat Fan D.-Y. , Jia H. , Barber J. , Chow W.S. ( 2009 ) Novel effects of methyl viologen on photosystem II function in spinach leaves . Eur. Biophys. J. 39 : 191 – 199 . Google Scholar Crossref Search ADS PubMed WorldCat Fan D.-Y. , Nie Q. , Hope A.B. , Hillier W. , Pogson B.J. , Chow W.S. ( 2007 ) Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction . Photosynth. Res. 94 : 347 – 357 . Google Scholar Crossref Search ADS PubMed WorldCat Furbank R.T. , Walker D.A. ( 1985 ) Photosynthetic induction in C4 leaves . Planta 163 : 75 – 83 . Google Scholar Crossref Search ADS PubMed WorldCat Gómez R. , Carrillo N. , Morelli M.P. , Tula S. , Shahinnia F. , Hajirezaei M.-R. , et al. ( 2018 ) Faster photosynthetic induction in tobacco by expressing cyanobacterial flavodiiron proteins in chloroplasts . Photosynth. Res. 136 : 129 – 138 . Google Scholar Crossref Search ADS PubMed WorldCat Hammond E.T. , Andrews T.J. , Mott K.A. , Woodrow I.E. ( 1998 ) Regulation of Rubisco activation in antisense plants of tobacco containing reduced levels of Rubisco activase . Plant J . 14 : 101 – 110 . Google Scholar Crossref Search ADS PubMed WorldCat Hendrickson L. , Furbank R.T. , Chow W.S. ( 2004 ) A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence . Photosynth. Res . 82 : 73 – 81 . Google Scholar Crossref Search ADS PubMed WorldCat Jia H. , Dwyer S.A. , Fan D.-Y. , Han Y. , Badger M.R. , von Caemmerer S. , et al. ( 2014 ) A novel P700 redox kinetics probe for rapid, non-intrusive and whole-tissue determination of Photosystem II functionality and the stoichiometry of the two photosystems in vivo . Physiol. Plant. 152 : 403 – 413 . Google Scholar Crossref Search ADS PubMed WorldCat Johnson G.N. ( 2011 ) Physiology of PSI cyclic electron transport in higher plants . Biochim. Biophys. Acta 1807 : 906 – 911 . Google Scholar Crossref Search ADS PubMed WorldCat Kaiser E. , Kromdijk J. , Harbinson J. , Heuvelink E. , Marcelis L.F.M. ( 2017 ) Photosynthetic induction and its diffusional, carboxylation and electron transport processes as affected by CO2 partial pressure, temperature, air humidity and blue irradiance . Ann. Bot. 119 : 191 – 205 . Google Scholar Crossref Search ADS PubMed WorldCat Kaiser E. , Morales A. , Harbinson J. , Kromdijk J. , Heuvelink E. , Marcelis L.F.M. ( 2015 ) Dynamic photosynthesis in different environmental conditions . J. Exp. Bot . 66 : 2415 – 2426 . Google Scholar Crossref Search ADS PubMed WorldCat Keren N. , Berg A. , van Kan P.J.M. , Levanon H. , Ohad I. ( 1997 ) Mechanism of Photosystem II photoinactivation and D1 protein degradation at low light: the role of back electron flow . Proc. Natl. Acad. Sci. USA 94 : 1579 – 1584 . Google Scholar Crossref Search ADS WorldCat Keren N. , Gong H. , Ohad I. ( 1995 ) Oscillations of reaction center II-D1 degradation in vivo induced by repetitive light flashes . J. Biol. Chem. 270 : 806 – 814 . Google Scholar Crossref Search ADS PubMed WorldCat Klughammer C. , Schreiber U. ( 2007 ) Saturation Pulse Method for Assessment of Energy Conversion in PS I. http://www.walz.com/downloads/pan/PAN07002.pdf (July 17, 2019, date last accessed). Klughammer C. , Schreiber U. ( 2008 ) Complementary PS II Quantum Yields Calculated from Simple Fluorescence Parameters Measured by PAM Fluorometry and the Saturation Pulse Method. http://www.walz.com/downloads/pan/PAN078007.pdf (July 17, 2019, date last accessed). Kou J. , Takahashi S. , Fan D.-Y. , Badger M.R. , Chow W.S. ( 2015 ) Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis . Front. Plant Sci . 6 : Article 758 . Google Scholar Crossref Search ADS PubMed WorldCat Kou J. , Takahashi S. , Oguchi R. , Fan D.-Y. , Badger M.R. , Chow W.S. ( 2013 ) Estimation of the steady-state cyclic electron flux around Photosystem I in spinach leaf discs in white light, CO2-enriched air and other varied conditions . Funct. Plant Biol. 40 : 1018 – 1028 . Google Scholar Crossref Search ADS WorldCat Kromdijk J. , Głowacka K. , Leonelli L. , Gabilly S.T. , Iwai M. , Niyogi K.K. , et al. ( 2016 ) Improving photosynthesis and crop productivity by accelerating recovery from photoprotection . Science 354 : 857 – 861 . Google Scholar Crossref Search ADS PubMed WorldCat Kroon B.M.A. , Thoms S. ( 2006 ) From electron to biomass: a mechanistic model to describe phytoplankton photosynthesis and steady-state growth rates . J. Phycol. 42 : 593 – 609 . Google Scholar Crossref Search ADS WorldCat Labs M. , Rühle T. , Leister D. ( 2016 ) The antimycin A-sensitive pathway of cyclic electron flow: from 1963 to 2015 . Photosyn. Res. 129 : 231 – 238 . Google Scholar Crossref Search ADS PubMed WorldCat Lennon A.M. , Prommeenate P. , Nixon P. ( 2003 ) Location, expression and orientation of the putative chlororespiratory enzymes, Ndh and IMMUTANS, in higher-plant plastids . Planta 218 : 254 – 260 . Google Scholar Crossref Search ADS PubMed WorldCat Martin M. , Noarbe D.M. , Serrot P.H. , Sabater B. ( 2015 ) The rise of the photosynthetic rate when light intensity increases is delayed in ndh gene-defective tobacco at high but not at low CO2 concentration . Front. Plant Sci . 6 : Article 34 . WorldCat Maxwell K. , Badger M.R. , Osmond C.B. ( 1998 ) A comparison of CO2 and O2 exchange patterns and the relationship with chlorophyll fluorescence during photosynthesis in C3 and CAM plants . Funct. Plant Biol. 25 : 45 – 52 . Google Scholar Crossref Search ADS WorldCat Mekala N.R. , Suorsa M. , Rantala M. , Aro E.-M. , Tikkanen M. ( 2015 ) Plants actively avoid state transitions upon changes in light intensity: role of light-harvesting complex II protein dephosphorylation in high light . Plant Physiol. 168 : 721 – 734 . Google Scholar Crossref Search ADS PubMed WorldCat Mi H. , Endo T. , Schreiber U. , Ogawa T. , Asada K. ( 1994 ) NAD(P)H dehydrogenase-dependent cyclic electron flow around photosystem I in the cyanobacterium Synechococystis PCC 6803: a study of dark-starved cells and spheroplasts . Plant Cell Physiol . 35 : 163 – 173 . WorldCat Miyake C. , Shinzaki Y. , Miyata M. , Tomizawa K-I. ( 2004 ) Enhancement of cyclic electron flow around PSI at high light and its contribution to the induction of non-photochemical quenching of Chl fluorescence in intact leaves of tobacco plants . Plant Cell Physiol . 45 : 1426 – 1433 . Google Scholar Crossref Search ADS PubMed WorldCat Munekage Y. , Hojo M. , Meurer J. , Endo T. , Tasaka M. , Shikanai T. ( 2002 ) PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis . Cell 110 : 361 – 371 . Google Scholar Crossref Search ADS PubMed WorldCat Osterhout W.J.V. , Haas A.R.C. ( 1918 ) Dynamic aspects of photosynthesis . Proc. Natl. Acad. Sci. USA 4 : 85 – 91 . Google Scholar Crossref Search ADS WorldCat Pearcy R.W. ( 1990 ) Sunflecks and photosynthesis in plant canopies . Annu. Rev. Plant Physiol. Plant Mol. Biol. 41 : 421 – 453 . Google Scholar Crossref Search ADS WorldCat Pearcy R.W. , Way D.A. ( 2012 ) Two decades of sunfleck research: looking back to move forward . Tree Physiol. 32 : 1059 – 1061 . Google Scholar Crossref Search ADS PubMed WorldCat Peltier G. , Tolleter D. , Billon E. , Cournac L. ( 2010 ) Auxiliary electron transport pathways in chloroplasts of microalgae . Photosynth. Res. 106 : 19 – 31 . Google Scholar Crossref Search ADS PubMed WorldCat Prinsley R.T. , Leegood R.C. ( 1986 ) Factors affecting photosynthetic induction in spinach leaves . Biochim. Biophys. Acta 849 : 244 – 253 . Google Scholar Crossref Search ADS WorldCat Rozak P.R. , Seiser R.M. , Wacholtz W.F. , Wise R.R. ( 2002 ) Rapid, reversible alterations in spinach thylakoid appression upon changes in light intensity . Plant. Cell Environ. 25 : 421 – 429 . Google Scholar Crossref Search ADS WorldCat Rubin B.T. , Barber J. , Paillotin G. , Chow W.S. , Yamamoto Y. ( 1981 ) A diffusional analysis of the temperature sensitivity of the Mg2+-induced rise of chlorophyll fluorescence . Biochim. Biophys. Acta 638 : 69 – 74 . Google Scholar Crossref Search ADS WorldCat Satoh K. , Strasser R. , Butler W.L. ( 1976 ) A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts . Biochim. Biophys. Acta. 440 : 337 – 345 . Google Scholar Crossref Search ADS PubMed WorldCat Shaku K. , Shimakawa G. , Hashiguchi M. , Miyake C. ( 2015 ) Reduction-induced suppression of electron flow (RISE) in the photosynthetic electron transport system of Synechococcus elongatus PCC 7942 . Plant Cell Physiol . 57 : 1443 – 1453 . Google Scholar PubMed WorldCat Shen Y.-K. , Chow W.S. , Park Y.-I. , Anderson J.M. ( 1996 ) Photoinactivation of photosystem II by cumulative exposure to short light pulses during the induction period of photosynthesis . Photosynth. Res. 47 : 51 – 59 . Google Scholar Crossref Search ADS PubMed WorldCat Shimakawa G. , Shaku K. , Miyake C. ( 2018 ) Reduction-induced suppression of electron flow (RISE) is relieved by non-ATP-consuming electron flow in Synechococcus elongatus PCC 7942 . Front. Microbiol . 9 : Article 886. WorldCat Shubin V.V. , Terekhova I.N. , Kirillov B.A. , Karapetyan N.V. ( 2008 ) Quantum yield of P700+ photodestruction in isolated photosystem I complexes of the cyanobacterium Arthrospira platensis . Photochem. Photobiol. Sci. 7 : 956 – 962 . Google Scholar Crossref Search ADS PubMed WorldCat Soleh M.A. , Tanaka Y. , Nomoto Y. , Iwahashi Y. , Nakashima K. , Fukuda Y. , et al. ( 2016 ) Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycin max (L.) Merr] . Plant Cell Environ . 39 : 685 – 693 . Google Scholar Crossref Search ADS PubMed WorldCat Stepien P. , Johnson G.N. ( 2009 ) Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal oxidase as an alternative electron sink . Plant Physiol. 149 : 1154 – 1165 . Google Scholar Crossref Search ADS PubMed WorldCat Stepien P. , Johnson G.N. ( 2018 ) Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport . Proc. Natl. Acad. Sci. USA 115 : 9634 – 9639 . Google Scholar Crossref Search ADS WorldCat Strand D.D. , Fisher N. , Kramer D.M. ( 2017 ) The higher plant plastid NAD(P)H dehydrogenase-like complex (NDH) is a high efficiency proton pump that increases ATP production by cyclic electron flow . J. Biol. Chem. 292 : 11850 – 11860 . Google Scholar Crossref Search ADS PubMed WorldCat Tagawa K. , Tsujimoto H.Y. , Arnon D.I. ( 1963 ) Role of chloroplast ferredoxin in the energy conservation process of photosynthesis . Proc. Natl. Acad. Sci. USA 49 : 567 – 572 . Google Scholar Crossref Search ADS WorldCat Takagi D. , Ifuku K. , Nishimura T. , Miyake C. ( 2018 ) Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II . Photosynth. Res . 139 : 487 – 498 . Google Scholar Crossref Search ADS PubMed WorldCat Takahashi S. , Milward S.E. , Fan D.-Y. , Chow W.S. , Badger M.R. ( 2009 ) How does cyclic electron flow alleviate photoinhibition in Arabidopsis? Plant Physiol. 149 : 1560 – 1567 . Google Scholar Crossref Search ADS PubMed WorldCat Taylor S.H. , Long S.P. ( 2017 ) Slow induction of photosynthesis on shade to sun transitions in wheat may cost at least 21% of productivity . Phil. Trans. R. Soc B 372 : 20160543 . Google Scholar Crossref Search ADS WorldCat Tikhonov A.N. ( 2015 ) Induction events and short-term regulation of electron transport in chloroplasts: an overview . Photosynth. Res. 125 : 65 – 94 . Google Scholar Crossref Search ADS PubMed WorldCat Tikkanen M. , Grieco M. , Kangasjärvi S. , Aro E.-M. ( 2010 ) Thylakoid protein phosphorylation in higher plant chloroplasts optimizes electron transfer under fluctuating light . Plant Physiol. 152 : 723 – 735 . Google Scholar Crossref Search ADS PubMed WorldCat Trissl H.-W. , Lavergne J. ( 1995 ) Fluorescence induction from Photosystem II: analytical equations for the yields of photochemistry and fluorescence derived from analysis of a model including exciton-radical pair equilibrium and restricted transfer between photosynthetic units . Funct. Plant Biol. 22 : 183 – 193 . Google Scholar Crossref Search ADS WorldCat Vass I. ( 2011 ) Role of charge recombination processes in photodamage and photoprotection of the photosystem II complex . Physiol. Plant. 142 : 6 – 16 . Google Scholar Crossref Search ADS PubMed WorldCat Vredenberg W.J. , Bulychev A.A. ( 2010 ) Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700+ induction kinetics . Biochim. Biophys. Acta 1797 : 1521 – 1532 . Google Scholar Crossref Search ADS PubMed WorldCat Walker D.A. ( 1973 ) Photosynthetic induction phenomena and the light activation of ribulose diphosphate carboxylase . New Phytol. 72 : 209 – 235 . Google Scholar Crossref Search ADS WorldCat Wood W.H.J. , MacGregor-Chatwin C. , Barnett S.F.H. , Mayneord G.E. , Huang X. , Hobbs J.K. , et al. ( 2018 ) Dynamic thylakoid stacking regulates the balance between linear and cyclic photosynthetic electron transfer . Nature Plants 4 : 116 – 127 . Google Scholar Crossref Search ADS PubMed WorldCat Xu D.-Q. , Gifford R.M. , Chow W.S. ( 1992 ) Effect of fusicoccin on photosynthetic induction in soybean leaves . Photosynthetica 26 : 770 – 773 . WorldCat Zhang M.-M. , Fan D.-Y. , Sun G.-Y. , Chow W.S. ( 2018 ) Optimizing the linear electron transport rate measured by chlorophyll a fluorescence to empirically match the gross rate of oxygen evolution in white light: towards improved estimation of the cyclic electron flux around Photosystem I in leaves . Funct. Plant Biol. 45 : 1138 – 1148 . Google Scholar Crossref Search ADS WorldCat Zhang Y. , Kaiser E. , Zhang Y. , Yang Q. , Li T. ( 2019 ) Red/blue light ratio strongly affects steady-state photosynthesis, but hardly affects photosynthetic induction, in tomato (Solanum lycopersicum) . Physiol. Plant . 167 : 144 – 158 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes M.-M. Zhang and D.-Y. Fan contributed equally in this study and they should be considered equal first authors © The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Partially Dissecting Electron Fluxes in Both Photosystems in Spinach Leaf Disks during Photosynthetic Induction
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcz114
DA - 2019-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/partially-dissecting-electron-fluxes-in-both-photosystems-in-spinach-qqGJFF3fYI
SP - 2206
VL - 60
IS - 10
DP - DeepDyve
ER -