Stromal Reductants Research Articles

Three phases of energy-dependent induction of $${\text{P}}_{{700}}^{+}$$ P 700 + and Chl a fluorescence in Tradescantia fluminensis leaves

In plants, the short-term regulation (STR, seconds to minute time scale) of photosynthetic apparatus is associated with the energy-dependent control in the chloroplast electron transport, the distribution of light energy between photosystems (PS) II and I, activation/deactivation of the Calvin–Benson cycle (CBC) enzymes, and relocation of chloroplasts within the plant cell. In this work, using a dual-PAM technique for measuring the time-courses of P700 photooxidation and Chl a fluorescence, we have investigated the STR events in Tradescantia fluminensis leaves. The comparison of Chl a fluorescence and \({\text{P}}_{{700}}^{+}\) induction allowed us to investigate the contribution of the trans-thylakoid pH difference (ΔpH) to the STR events. Two parameters were used as the indicators of ΔpH generation: pH-dependent component of non-photochemical quenching of Chl a fluorescence, and pHin-dependent rate of electron transfer from plastoquinol (PQH2) to \({\text{P}}_{{700}}^{+}\) (via the Cyt b6f complex and plastocyanin). In dark-adapted leaves, kinetics of \({\text{P}}_{{700}}^{+}\) induction revealed three phases. Initial phase is characterized by rapid electron flow to \({\text{P}}_{{700}}^{+}\) (τ1/2 ~ 5–10 ms), which is likely related to cyclic electron flow around PSI, while the outflow of electrons from PSI is restricted by slow consumption of NADPH in the CBC. The light-induced generation of ΔpH and activation of the CBC promote photooxidation of P700 and concomitant retardation of \({\text{P}}_{{700}}^{+}\) reduction (τ1/2 ~ 20 ms). Prolonged illumination induces additional slowing down of electron transfer to \({\text{P}}_{{700}}^{+}\) (τ1/2 ≥ 30–35 ms). The latter effect is not accompanied by changes in the Chl a fluorescence parameters which are sensitive to ΔpH generation. We suggest the tentative explanation of the latter results by the reversal of Q-cycle, which causes the deceleration of PQH2 oxidation due to the back pressure of stromal reductants.

Linking chloroplast relocation to different responses of photosynthesis to blue and red radiation in low and high light-acclimated leaves of Arabidopsis thaliana (L.).

Low light (LL) and high light (HL)-acclimated plants of A. thaliana were exposed to blue (BB) or red (RR) light or to a mixture of blue and red light (BR) of incrementally increasing intensities. The light response of photosystem II was measured by pulse amplitude-modulated chlorophyll fluorescence and that of photosystem I by near infrared difference spectroscopy. The LL but not HL leaves exhibited blue light-specific responses which were assigned to relocation of chloroplasts from the dark to the light-avoidance arrangement. Blue light (BB and BR) decreased the minimum fluorescence ([Formula: see text]) more than RR light. This extra reduction of the [Formula: see text] was stronger than theoretically predicted for [Formula: see text] quenching by energy dissipation but actual measurement and theory agreed in RR treatments. The extra [Formula: see text] reduction was assigned to decreased light absorption of chloroplasts in the avoidance position. A maximum reduction of 30% was calculated. Increasing intensities of blue light affected the fluorescence parameters NPQ and qP to a lesser degree than red light. After correcting for the optical effects of chloroplast relocation, the NPQ responded similarly to blue and red light. The same correction method diminished the color-specific variations in qP but did not abolish it; thus strongly indicating the presence of another blue light effect which also moderates excitation pressure in PSII but cannot be ascribed to absorption variations. Only after RR exposure, a post-illumination overshoot of [Formula: see text] and fast oxidation of PSI electron acceptors occurred, thus, suggesting an electron flow from stromal reductants to the plastoquinone pool.

Formation mechanisms of superoxide radical and hydrogen peroxide in chloroplasts, and factors determining the signalling by hydrogen peroxide.

Reduction of O2 molecule to superoxide radical, O2•-, in the photosynthetic electron transport chain is the first step of hydrogen peroxide, H2O2, production in chloroplasts in the light. The mechanisms of O2 reduction by ferredoxin, by the components of the plastoquinone pool, and by the electron transfer cofactors in PSI are analysed. The data indicating that O2•- and H2O2 can be produced both outside and within thylakoid membrane are presented. The H2O2 production in the chloroplast stroma is described as a result of either dismutation of O2•- or its reduction by stromal reductants. Formation of H2O2 within thylakoid membrane in the reaction of O2•- with plastohydroquinone is examined. The significance of both ways of H2O2 formation for specificity of the signal being sent by photosynthetic electron transport chain to cell adaptation systems is discussed.

NADPH from the oxidative pentose phosphate pathway drives the operation of cyclic electron flow around photosystem I in high-intertidal macroalgae under severe salt stress.

Pyropia yezoensis (Bangiales, Rhodophyta) is a representative species of high-intertidal macroalgae, whose blades can tolerate extreme stresses, such as salt stress and desiccation. In this study, the photosystem (PS) responses of P. yezoensis blades under salt stress were studied. Our results showed that when the effective photochemical quantum yield of PS (Y) II decreased to almost zero under high salt stress, YI still had a relatively high activity rate. PSII was therefore more sensitive to salt stress than PSI. Furthermore, in the presence of 3-(3', 4'-dichlorophenyl)-1,1-dimethylurea (DCMU), YI rose as salinity increased. The YI values for DCMU-treated thalli decreased in the presence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PDH) inhibitor (glucosamine, Glucm). The YI values were ∼0.09 in the presence of methyl viologen (MV) and almost zero in the presence of dibromothymoquinone (DBMIB). These results demonstrated that under severe salt stress (120‰ salinity) PSI activity was driven from a source other than PSII, and that stromal reductants probably supported the operation of PSI. Under salt stress, the starch content decreased and soluble sugar levels increased. The G6PDH and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) activities increased, but cytosolic glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) activity decreased. Furthermore, the NADPH content increased, but NADH decreased, which suggested that soluble sugar entered the oxidative pentose phosphate pathway (OPPP). All these results suggested that NADPH from OPPP increases the cyclic electron flow around PSI in high-intertidal macroalgae under severe salt stress.

Specific photosynthetic and morphological characteristics allow macroalgae Gloiopeltis furcata (Rhodophyta) to survive in unfavorable conditions

Gloiopeltis furcata (Postels & Ruprecht) J. Agardh, a macroalga, which grows in an upper, intertidal zone, can withstand drastic environmental changes caused by the periodic tides. In this study, the photosynthetic and morphological characteristics of G. furcata were investigated. The photosynthetic performance and electron flows of the thalli showed significant variations in response to desiccation and salinity compared with the control group. Both PSII and PSI activities declined gradually when the thalli were under stress. However, the electron transport rate of PSI showed still a low value during severe conditions, while the rate of PSII approached zero. Furthermore, PSI activity of the treated thalli recovered faster than PSII after being submerged in seawater. Even though the linear electron flow was inhibited by DCMU [3-(3, 4-dichlorophenyl)-1,1-dimethylurea], the cyclic electron flow could still be restored. The rate of cyclic electron flow recovery declined with the increasing time of dark treatment, which suggested that stromal reductants from starch degradation played an important role in the donation of electrons to PSI. This study demonstrated that PSII was more sensitive than PSI to desiccation and salinity in G. furcata and that the cyclic electron flow around PSI played a significant physiological role. In addition, G. furcata had branches, which were hollow inside and contained considerable quantities of funoran. These might be the most important factors in allowing G. furcata to adapt to adverse intertidal environments.

How does iron deficiency disrupt the electron flow in photosystem I of lettuce leaves?

The changes observed photosystem I activity of lettuce plants exposed to iron deficiency were investigated. Photooxidation/reduction kinetics of P700 monitored as ΔA820 in the presence and absence of electron transport inhibitors and acceptors demonstrated that deprivation in iron decreased the population of active photo-oxidizable P700. In the complete absence of iron, the addition of plant inhibitors (DCMU and MV) could not recover the full PSI activity owing to the abolition of a part of P700 centers. In leaves with total iron deprivation (0μM Fe), only 15% of photo-oxidizable P700 remained. In addition, iron deficiency appeared to affect the pool size of NADP+ as shown by the decline in the magnitude of the first phase of the photooxidation kinetics of P700 by FR-light. Concomitantly, chlorophyll content gradually declined with the iron concentration added to culture medium. In addition, pronounced changes were found in chlorophyll fluorescence spectra. Also, the global fluorescence intensity was affected. The above changes led to an increased rate of cyclic electron transport around PSI mainly supported by stromal reductants.

The enhancement of cyclic electron flow around photosystem I improves the recovery of severely desiccated Porphyra yezoensis (Bangiales, Rhodophyta)

Porphyra yezoensis, a representative species of intertidal macro-algae, is able to withstand periodic desiccation at low tide but is submerged in seawater at high tide. In this study, changes in photosynthetic electron flow in P. yezoensis during desiccation and re-hydration were investigated. The results suggested that the cyclic electron flow around photosystem I (PSI) increased significantly during desiccation, continued to operate at times of severe desiccation, and showed greater tolerance to desiccation than the electron flow around PSII. In addition, PSI activity in desiccated blades recovered faster than PSII activity during re-hydration. Even though linear electron flow was suppressed by DCMU [3-(3',4'-dichlorophenyl)-1,1-dimethylurea], cyclic electron flow could still be restored. This process was insensitive to antimycin A and could be suppressed by dibromothymoquinone (DBMIB). The prolonged dark treatment of blades reduced the speed in which the cyclic electron flow around PSI recovered, suggesting that stromal reductants, including NAD(P)H, played an important role in the donation of electrons to PSI and were the main cause of the rapid recovery of cyclic electron flow in desiccated blades during re-hydration. These results suggested that cyclic electron flow in P. yezoensis played a significant physiological role during desiccation and re-hydration and may be one of the most important factors allowing P. yezoensis blades to adapt to intertidal environments.

Afterglow Thermoluminescence Measured in Isolated Chloroplasts

The thermoluminescence afterglow (AG) measured in plant leaves originates from the S(2)/S(3)Q(B)(-) charge pair recombination in photosystem II (PSII) initiated by reverse electron flow from stromal reductants to PQ and then to the Q(B) site in PSII centers that are in the S(2)/S(3)Q(B) state. In this study, we show that this luminescence, absent in isolated thylakoid membranes, can be measured in intact chloroplasts that retain their stromal content including the electron acceptor pool (oxidized ferredoxin/NADP(+)) of photosystem I. The properties of the chloroplasts AG emission is similar to the AG in leaves in terms of temperature maximum, period-four modulation, far-red light stimulation, and antimycin A inhibition.

Structure of the Chloroplast NADH Dehydrogenase-Like Complex: Nomenclature for Nuclear-Encoded Subunits

The chloroplast NADH dehydrogenase-like complex (NDH) was first discovered based on its similarity to complex I in respiratory electron transport, and is involved in electron transport from photoproduced stromal reductants such as NADPH and ferredoxin to the intersystem plastoqunone pool. However, a recent study suggested that it is a ferredoxin-dependent plastoquinone reductase rather than an NAD(P)H dehydrogenase. Furthermore, recent advances in subunit analysis of NDH have revealed the presence of a novel hydrophilic subcomplex on the stromal side of the thylakoid membrane, as well as an unexpected lumenal subcomplex. This review discusses these new studies on the structure of NDH, and proposes a unified nomenclature for newly discovered NDH subunits.

Equilibrium or disequilibrium? A dual-wavelength investigation of photosystem I donors

Oxidation of photosystem I (PSI) donors under far-red light (FRL), slow re-reduction by stromal reductants and fast re-reduction in the dark subsequent to illumination by white light (WL) were recorded in leaves of several C(3) plants at 810 and 950 nm. During the re-reduction from stromal reductants the mutual interdependence of the two signals followed the theoretical relationship calculated assuming redox equilibrium between plastocyanin (PC) and P700, with the equilibrium constant of 40 +/- 10 (Delta E (m) = 86-99 mV) in most of the measured 24 leaves of nine plant species. The presence of non-oxidizable PC of up to 13% of the whole pool, indicating partial control of electron transport by PC diffusion, was transiently detected during a saturation pulse of white light superimposed on FRL or on low WL. Nevertheless, non-oxidizable PC was absent in the steady state during fast light-saturated photosynthesis. It is concluded that in leaves during steady state photosynthesis the electron transport rate is not critically limited by PC diffusion, but the high-potential electron carriers PC and P700 remain close to the redox equilibrium.

A qualitative analysis of the regulation of cyclic electron flow around photosystem I from the post-illumination chlorophyll fluorescence transient in Arabidopsis: a new platform for the in vivo investigation of the chloroplast redox state

A transient in chlorophyll fluorescence after cessation of actinic light illumination, which has been ascribed to electron donation from stromal reductants to plastoquinone (PQ) by the NAD(P)H-dehydrogenase (NDH) complex, was investigated in Arabidopsis thaliana. The transient was absent in air in a mutant lacking the NDH complex (ndhM). However, in ndhM, the transient was detected in CO(2)-free air containing 2% O(2). To investigate the reason, ndhM was crossed with a pgr5 mutant impaired in ferredoxin (Fd)-dependent electron donation from NADPH to PQ, which is known to be redundant for NDH-dependent PQ reduction in the cyclic electron flow around photosystem I (PSI). In ndhM pgr5, the transient was absent even in CO(2)-free air with 2% O(2), demonstrating that the post-illumination transient can also be induced by the Fd- (or PGR5)-dependent PQ reduction. On the other hand, the transient increase in chlorophyll fluorescence was found to be enhanced in normal air in a mutant impaired in plastid fructose-1,6-bisphosphate aldolase (FBA) activity. The mutant, termed fba3-1, offers unique opportunities to examine the relative contribution of the two paths, i.e., the NDH- and Fd- (or PGR5)-dependent paths, on the PSI cyclic electron flow. Crossing fba3-1 with either ndhM or pgr5 and assessing the transient suggested that the main route for the PSI cyclic electron flow shifts from the NDH-dependent path to the Fd-dependent path in response to sink limitation of linear electron flow.

A Novel Nucleus-Encoded Chloroplast Protein, PIFI, Is Involved in NAD(P)H Dehydrogenase Complex-Mediated Chlororespiratory Electron Transport in Arabidopsis

A transient rise in chlorophyll fluorescence after turning off actinic light reflects nonphotochemical reduction of the plastoquinone (PQ) pool. This process is dependent on the activity of the chloroplast NAD(P)H dehydrogenase (NDH) complex, which mediates electron flow from stromal reductants to the PQ pool. In this study, we characterized an Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant pifi (for postillumination chlorophyll fluorescence increase), which possesses an intact NDH complex, but lacks the NDH-dependent chlorophyll fluorescence increase after turning off actinic light. The nuclear gene PIFI (At3g15840) containing the T-DNA insertion encodes a chloroplast-targeted protein localized in the stroma and is annotated as a protein of unknown function. The pifi mutant exhibited a lower capacity for nonphotochemical quenching, but similar CO(2) assimilation rates, photosystem II (PSII) quantum efficiencies (PhiPSII), and reduction levels of the primary electron acceptor of PSII (1 - qL) as compared with the wild type. The pifi mutant grows normally under optimal conditions, but exhibits greater sensitivity to photoinhibition and long-term mild heat stress than wild-type plants, which is consistent with lower capacity of nonphotochemical quenching. We conclude that PIFI is a novel component essential for NDH-mediated nonphotochemical reduction of the PQ pool in chlororespiratory electron transport.

Nonphotosynthetic Reduction of the Intersystem Electron Transport Chain of Chloroplasts Following Heat Stress. The Pool Size of Stromal Reductants¶

The properties of a negative transient signal (negative peak) observed during the first seconds of the induction of the photoacoustic (PA) signal in dark-adapted barley leaves treated with methyl viologen (MV) and diuron and then exposed to high temperatures have been examined. Under those conditions no electron donation from photosystem II (PSII) occurred, and electron flow through PSI could be supported only by soluble reductants located in the chloroplast stroma. The negative peak was observed only if the PA signal had been monitored at low, and not high, frequencies. The peak obviously originated from the oxygen consumption by PSI. The size of the peak increased as the temperature of preheating was raised from 39 to 45°C. The size of the peak decreased exponentially with a half-time of 3.7 s during illumination under low light. This decrease was found to be much faster under strong light. The recovery of the peak during dark acclimation required several minutes. It is concluded that the negative peak reflects the oxygen consumption supported by stromal reductants, their pool being rapidly exhausted under light in the presence of MV. The maximal size of the pool was calculated as 140 eq:P700 in dark-adapted leaves.

Effect of exogenous glucose on electron flow to photosystem I and respiration in cyanobacterial cells

The effects of exogenous glucose on the rates of alternative pathways of photosystem II (PSII)-independent electron flow to PSI and of dark respiration in Synechocystis sp. 6803 cells were studied. The presence of glucose was shown to accelerate the electron flow to P700+, the PSI primary electron donor oxidized with Far-red light (FRL), which excites specifically only PSI. An increase in the glucose concentration was accompanied by a further activation of electron flow to PSI, which was supported by the dark donation of reducing equivalents to the electron transport chain. An increase in the external glucose concentration resulted also in the disappearance of lag-phase in the kinetics of P700+ reduction, which was observed in the cells incubated without glucose after FRL switching off. A similarity of nonphotochemical processes of electron transfer to PSI in cyanobacteria and higher plants was supposed, basing on the earlier observed fact of the occurrence of such lagphase in higher plants and its dependence on the exhausting of stromal reductants in the light. Acceleration of dark electron flow to PSI in the presence of glucose, a major respiratory substrate, may indicate the coupling between nonphotochemical processes in the photosynthetic and respiratory chains of electron transport in cyanobacterial cells. A close correlation between photosynthesis and respiration in cyanobacterial cells is also confirmed by a sharp acceleration of respiration with an increase in the glucose concentration in medium.

Modulating Effect of Far-Red Light on Activities of Alternative Electron Transport Pathways Related to Photosystem I

Barley (Hordeum vulgare L.) leaves were irradiated with far-red (FR) light of various intensities after different periods of dark adaptation in order to investigate activities of alternative electron transport pathways related to photosystem I (PSI). Photooxidation of P700, the primary electron donor of PSI, was saturated at FR light intensity of 0.15 µmol quanta/(m2 s). As the photon flux density was raised in this range, the slow and middle components in the kinetics of P700+ dark reduction increased, whereas the fast component remained indiscernible. The amplitudes of the slow and middle components diminished upon further increase of FR photon flux density in the range 0.15–0.35 µmol quanta/(m2 s) and remained constant at higher intensities. The fast component of P700+ reduction was only detected after FR irradiation with intensities above 0.15 µmol quanta/(m2 s); the light-response curve for this component was clearly sigmoid. In dark-adapted barley leaves, three stages were distinguished in the kinetics of P700 photooxidation, with the steady state for P700+ achieved within about 3 min. In leaves predarkened for a short time, the onset of FR irradiation produced a very rapid photooxidation of P700. As the duration of dark exposure was prolonged, the amplitude of the first peak in the kinetic curve of photoinduced P700 photooxidation was diminished and the time for attaining the steady-state oxidation level was shortened. After a brief dark adaptation of leaves, ferredoxin-dependent electron flow did not appreciably contributed to the kinetics of P700+ dark reduction, whereas the components related to electron donation from stromal reductants were strongly retarded. It is concluded that FR light irradiation, selectively exciting PSI, suffices to modulate activities of alternative electron transport routes; this modulation reflects the depletion of stromal reductants due to continuous efflux of electrons from PSI to oxygen under the action of FR light.

Elevated Temperatures Inhibit Ferredoxin-Dependent Cyclic Electron Flow around Photosystem I

In experiments with barley (Hordeum vulgare L.) leaves, absorbance changes at 830 nm induced by far-red light were measured as indicator of redox conversions of primary electron donor (P700) of photosystem I (PSI). Using this method, the action of elevated temperature (45°C, 5 min) on PSI-driven electron transport through alternative pathways was examined. Thermally induced inactivation was found to transform nonmonotonic photooxidation of P700, induced by far-red light in untreated leaves, into a fast and monotonic process completed within 1-s illumination. The short-term heating of leaves fully eliminated the fast component in the kinetics of P700+ dark reduction, related to operation of ferredoxin-dependent cyclic electron transport around PSI. At the same time, thermoinactivation substantially accelerated the slow and middle components of dark P700+ reduction, i.e., the components determined by arrival of electrons to PSI from reductants located in the chloroplast stroma. The latter effect was also observed after heating of leaves pretreated with antimycin A or methyl viologen; both agents are known to inhibit the ferredoxin-dependent electron transport. It is concluded that the heat treatment of leaves inhibits the ferredoxin-dependent pathway of electron transport around PSI and activates electron transport through alternative routes providing reducing equivalents to PSI from stromal reductants.

Populations of photosystem 1 units rapidly and slowly reduced by stromal reductants represent photosystem 1α and photosystem 1β complexes: Evidence from irradiance-response curves of P700 photooxidation in intact barley leaves

Photon-induced absorbance changes at 830 nm (ΔA830) related to redox transformations of P700, primary electron donor of photosystem 1 (PS1), were examined in barley leaves treated with diuron and methyl viologen. In such leaves, only soluble reductants localized in chloroplast stroma could serve as electron donors for P700+. Δ A830 were induced by 1-min irradiation of leaves with “actinic light” (AL, 700±6 nm) of various irradiances. Two exponentially decaying components with half-times of 2.75 (fast component, relative magnitude of 62 % of ΔA830) and 11.90 s (slow one, 38 % of ΔA830) were distinguished in the kinetics of dark relaxation of ΔA830 after leaf irradiation with saturating AL. The components reflecting P700+ dark reduction in two units of PS1 differed in the rate of electron input from stromal reductants. The decline in AL irradiance reduced steady state δA830 magnitude, which was also accompanied by a decrease in the contribution of fast component to the overall P700+ dark reduction kinetics. The photon-response curves were obtained separately for rapidly and slowly decaying δA830. The values of half-saturating irradiance were 0.106 and 0.035 μmol m−2 s−1 for rapidly and slowly reduced PS1 units, respectively. The ratio of rate constants of P700+ dark reduction for rapidly and slowly reduced PS1 units was 1.4 times higher than the ratio of their half-saturating irradiances thus indicating higher relative antenna size in rapidly reduced PS1 units. The latter finding, taken together with higher relative amount of P700, favours the view that rapidly and slowly reduced PS1 units reflect P700+ reduction by stromal reductants in spatially separated PS1α and PS1β complexes.

Redox states of photosystems I and II in irradiated leaves of wheat seedlings grown under different conditions of nitrogen nutrition

Changes in the redox states of photosystem I (PSI) and PSII in irradiated wheat leaves were studied after growing seedlings on a nitrogen-free medium or media containing either nitrate or ammonium. The content of P700, the primary electron donor of PSI was quantified using the maximum magnitude of absorbance changes at 830 nm induced by saturating white light. The highest content of P700 in leaves was found for seedlings grown on the ammonium-containing medium, whereas its lowest content was observed on seedlings grown in the presence of nitrate. At all irradiances of actinic light, the smallest accumulation of reduced QA was observed in leaves of ammonium-grown plants. Despite variations in light-response curves of P700 photooxidation and QA photoreduction, the leaves of all plants exposed to different treatments demonstrated similar relationships between steady-state levels of P700+ and QA−. The accumulation of oxidized P700 up to 40% of total P700 content was not accompanied by significant QA photoreduction. At higher extents of P700 photooxidation, a linear relationship was found between the steady-state levels of P700+ and QA−. The leaves of all treatments demonstrated biphasic patterns of the kinetics of P700+ dark reduction after irradiation by far-red light exciting specifically PSI. The halftimes of corresponding kinetic components were found to be 2.6–4 s (fast component) and 17–22 s (slow component). The two components of P700+ dark reduction were related to the existence of two PSI populations with different rates of electron input from stromal reductants. The magnitudes of these components differed for plants grown in the presence of nitrate, on the one hand, and plants grown either in the presence of ammonium or in the absence of nitrogen, on the other hand. This indicates the possible influence of nitrogen nutrition on synthesis of different populations of PSI in wheat leaves. The decrease in far-red light irradiance reduced the relative contribution of the fast component to P700+ reduction. The fast component completely disappeared at low irradiances. This finding indicates that the saturating far-red light must be applied to determine correctly the relative content of each PSI population in wheat leaves.

Cyclic electron flow around PSI monitored by afterglow luminescence in leaves of maize inbred lines (Zea mays L.): correlation with chilling tolerance

Maize (Zea mays L.) inbred lines of contrasting chilling sensitivity (three tolerant, three sensitive lines) were acclimated to 280 mumol photons m(-2) s(-1) white light at a 17 degrees C sub-optimal temperature. They showed no symptoms of photoinhibition, despite slight changes in photosystem II (PSII) fluorescence and thermoluminescence properties in two tolerant lines. A luminescence "afterglow" emission [Bertsch and Azzi (1965) Biochim Biophys Acta 94:15-26], inducible by a far-red (FR) illumination of unfrozen leaf discs, was detected either as a bounce in decay kinetics at constant temperatures or as a sharp thermoluminescence afterglow band at about 45 degrees C, in dark-adapted leaves. This band reflects the induction by warming of an electron pathway from stromal reductants to plastoquinones and to the Q(B) secondary acceptor of PSII, resulting in a luminescence-emitting charge recombination in the fraction of centres that were initially in the S(2/3)Q(B) non-luminescent state. A 5-h exposure of plants to growth chamber light shifted this luminescence emission towards shorter times and lower temperatures for several hours in the three chilling-tolerant lines. This downshift was not observed, or only transiently, in the three sensitive lines. In darkness, the downshifted afterglow band relaxed within hours to resume its dark-adapted location, similar for all maize lines. A faster dark re-reduction of P700(+) oxidized by FR light (monitored by 820-nm absorbance) and an increase of photochemical energy storage under FR excitation (determined by photoacoustic spectroscopy) confirmed that a cyclic pathway induced by white actinic light remained activated for several hours in the tolerant maize lines.

Electron Fluxes through Photosystem I in Cucumber Leaf Discs Probed by far-red Light

Cucumber leaf discs were illuminated at room-temperature with far-red light to photo-oxidise P700, the chlorophyll dimer in Photosystem (PS) I. The post-illumination kinetics of P700(+) re-reduction were studied in the presence of inhibitors or cofactors of photosynthetic electron transport. The re-reduction kinetics of P700(+) were well fitted as the sum of three exponentials, each with its amplitude and rate coefficient, and an initial flux (at the instant of turning off far-red light) given as the product of the two. Each initial flux is assumed equal to a steady state flux during far-red illumination. The fast phase of re-reduction, with rate coefficient k (1) approximately 10 s(-1), was completely abolished by a saturating concentration of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); it is attributed to electron flow to P700(+) from PS II, which was stimulated to some extent by far-red light. The intermediate phase, with rate coefficient k (1) approximately 1 s(-1), was only partly diminished by methyl viologen (MV) which diverts electron flow to oxygen. The intermediate phase is attributed to electron donation from reduced ferredoxin to the intersystem pool; reduced ferredoxin could be formed: (1) directly by electron donation on the acceptor of PS I; and/or (2) indirectly by stromal reductants, in line with only a partial inhibition of the intermediate phase by MV. Duroquinol enhanced the intermediate phase in the presence of DCMU, presumably through its interaction with thylakoid membrane components leading to the partial reduction of plastoquinone. The slow phase of P700(+) re-reduction, with rate coefficient k (1) approximately 0.1 s(-1), was unaffected by DCMU and only slightly affected by MV; it could be associated with electron donation to either: (1) the intersystem chain by stromal reductants catalysed by NAD(P)H dehydrogenase slowly; or (2) plastocyanin/P700(+) by ascorbate diffusing across the thylakoid membrane to the lumen. It is concluded that a post-illumination analysis of the fluxes to P700(+) can be used to probe the pathways of electron flow to PS I in steady state illumination.