Intersystem Chain Research Articles

Manganese deficiency alters photosynthetic electron transport in Marchantia polymorpha

Manganese (Mn) is considered as an essential element for plant growth. Mn starvation has been shown to affect photosystem II, the site of the Mn4CaO5 cluster responsible for water oxidation. Less is known on the effect of Mn starvation on photosystem I. Here we studied the effects of Mn deficiency in vivo on redox changes of P700 and plastocyanin (Pc) in the liverwort Marchantia polymorpha using the KLAS-NIR spectrophotometer. Far-red illumination is used to excite preferentially photosystem I, thus facilitating cyclic electron transport. Under Mn starvation, we observed slower oxidation of P700 and a decrease in the Pc signal relative to P700. The lower Pc content under Mn deficiency was confirmed by western blots. Re-reduction kinetics of P700+ and Pc+ were faster in Mn deficient thalli than in the control. The above findings show that the kinetics studied under Mn deficiency not only depend on the number of available reductants but also on how quickly electrons are transferred from stromal donors via the intersystem chain to Pc+ and P700+. We suggest that under Mn deficiency a structural reorganization of the thylakoid membrane takes place favoring the formation of supercomplexes between ferredoxin, cytochrome b6f complex, Pc and photosystem I, and thus an enhanced cyclic electron transport.

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803.

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b6/f complex, plastocyanin, and cytochrome c6, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700+) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700+ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700+ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

Monitoring moderate Cu and Cd toxicity by chlorophyll fluorescence and P<sub>700</sub> absorbance in pea leaves

We investigated the effect of moderate Cu2+ and Cd2+ stress by applying chlorophyll (Chl) fluorescence and P700 absorbance measurements to monitor the photosynthetic electron transport activity of 3-week-old Pisum sativum L. cv. Petit Provencal plants grown in a modified Hoagland solution containing 50 μM CuSO4 or 5 μM CdCl2. Both heavy metals caused a slight inhibition in PSII photochemistry as indicated by the decrease in the effective quantum efficiency of PSII (ΦPSII), the maximum electron transport capacity (ETRmax), and the maximum quantum yield for electron transport (α). PSI photochemistry was also affected by these heavy metals. Cu2+ and Cd2+ decreased the quantum efficiency of PSI (ΦPSI) as well as the number of electrons in the intersystem chain, and the Cu2+ treatment significantly reduced the number of electrons from stromal donors available for PSI. These results indicate that PSII and PSI photochemistry of pea plants are both sensitive to moderate Cu2+ and Cd2+ stress, which in turn is easily detected and monitored by Chl fluorescence and P700 absorbance measurements. Therefore, monitoring the photochemistry of pea plants with these noninvasive, yet sensitive techniques offers a promising strategy to study heavy metal toxicity in the environment.

State Transitions in Chlamydomonas reinhardtii. The Role of the Mehler Reaction in State 2-to-State 1 Transition

The light intensity-dependent transition to state 1 of dark-adapted anaerobic state 2 Chlamydomonas reinhardtii cells is stimulated by oxygen and by other electron acceptors for photosystem I, such as oxaloacetate and methylviologen. This suggests that the transition to state 1 requires the oxidation of the intersystem chain by photosystem I photochemistry. On the other hand, the mere oxidation in the dark of the chain-by addition of O2-leads only to a slow and incomplete transition. The light-driven stimulation by O2 of the state 1 transition is saturated at an O2 concentration of 15 to 20 microm, definitely higher than that of respiration. We suggest that this may represent the affinity for oxygen of the Mehler reaction, a conclusion that is confirmed by the observations that mitochondrial respiration is apparently not involved in modulating state 2-to-state 1 transition. The catalysis of the state 2-to-state 1 transition upon illumination of anaerobically adapted algae might represent, therefore, a relevant physiological role of this process in C. reinhardtii.

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.

Thermal Sensitivity of the Pool Size of Electrons Available to P700<sup>+</sup> Reduction in Intact Maize Leaves

The relative size of the pool of electrons accumulated in stroma reductants during actinic irradiation, which can be donated to P700+via the intersystem chain, was estimated after short-term exposure of intact Zea mays leaves to elevated temperatures. When the temperature increased from 25 to 50 °C by 5 °C steps, the relative size of the stroma electron pool went through a maximum at around 30 °C, and decreased gradually thereafter.

Photo-induction of an NADPH dehydrogenase which functions as a mediator of electron transport to the intersystem chain in the cyanobacterium Synechocystis PCC6803.

Illumination of the dark-incubated cells of Synechocystis PCC6803 caused recovery of both respiratory activity of oxygen uptake and PS I-cyclic electron flow, which was monitored by the dark reduction of P700(+) in the presence of DCMU after a 50 ms pulse light (MT) under background far-red light, but the effects were much smaller in those of the mutant M55, which has an ndh-B defective gene. Activity of an NADPH-NBT oxidoreductase with a higher molecular mass (around 380 kDa), which was only found in wild type but not in M55, became evident after the dark-incubated cells were exposed to the light. Immuno-blotting analysis indicated that the NADPH-NBT oxidoreductase contains the NdhB subunit of NDH. The expression of NdhB decreased in dark-incubated cells and increased upon transfer of the cells back to light. These results indicate that an NADPH-specific NDH participates in the light-regulated cyclic electron transport around Photosystem I as well as in respiratory electron transport to the intersystem chain in Synechocystis 6803.

Stromal Electron flow through the plastoquinone poolcontrols the state transition Capacity in Sunechococcus sp. PCC 7942 cells. Effects of iron deficiency

Chlorophyll fluorescence measurements have indicated that dark-adapted cyanobacterial cells are usually in State II characterised by low PSII fluorescence yield. Upon illumination, the quenching of PSII was released and fluorescence increased to a much higher level characteristic for cells in State I. In contrast, iron deficient cells exhibited higher PSII fluorescence in dark-adapted state and no fluorescence changes upon illumination were registered. This indicates that the cells are locked in State I under iron deficient conditions. Low temperature (77K) fluorescence measurements confirmed this suggestion. Following the postilumination increase of Fo as a measure of the dark reduction of the PQ pool, it was found that a massive stromal electron flow exists in the control iron sufficient cells. On the contrary, no measurable dark reduction of the PQ pool was detected in iron deficient cells. Interestingly, preventing the HADP dehydrogenase-dependent stromal electron flow to the intersystem chain completely inhibited the light induced increase of PSII fluorescence in control cells. 77K measurements indicated that under these dark-adapted conditions, cells were shifted from State II to State I and the observed 77K fluorescence pattern resembled that of iron deficient cells. Based on these results, it is reasonable to suggest that the state transition phenomena in Synechococcus strongly depend on the redox state of the PQ pool and that the NADP-dependent sromal electron flow is one of the major mechanisms controlling it. Since the NADP-dehydrogenase is one of the most Fe-abundant complexes within the cyanobacterial thylakoid membranes minimal stromal electron flow should be expected. This would prevent the PQ pool from dark reduction and keep the iron deficient cells in state I.

Reduction of the plastoquinone pool by exogenous NADH and NADPH in higher plant chloroplasts: Characterization of a NAD(P)H–plastoquinone oxidoreductase activity

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching F max, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H–PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin–NADP +-reductase (FNR), we infer that FNR is not involved in the NAD(P)H–PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H–PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H–PQ oxidoreductase is estimated to about 160 nmol O 2 min −1 mg −1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes.

Effects of inorganic carbon accumulation on photosynthetic oxygen reduction and cyclic electron flow in the cyanobacterium Synechococcus PCC7942

This paper examines the effect of inorganic carbon transport and accumulation in Synechococcus PCC7942 on fluorescence quenching, photosynthetic oxygen reduction and both linear and cyclic electron flow. The data presented support the previous findings of Miller et al. (1991) that the accumulation of Ci by the CO2 concentrating mechanism is able to stimulate oxygen photoreduction, particularly so when CO2 fixation is inhibited by PCR cycle inhibitors such as glycolaldehyde. This effect is found with both high and low-Ci grown cells, but the potential for oxygen photoreduction is about two-fold higher in low-Ci grown cells. This greater potential for O2 photoreduction is also correlated with a higher ability of low-Ci cells to photoreduce H2O2. Experiments with a mutant which transports Ci but does not accumulate it internally, indicates that the stimulation of O2 photoreduction appears to be a direct effect of the internal accumulation of Ci rather than from its participation in the transport process. In the absence of Ci, no specific partial reactions of photosynthetic electron transport appear to be inhibited, and the PS 1 acceptors PNDA and MV as well as the PS 2 acceptor DMQ can all run electron transport at levels approaching those during active CO2 fixation. Measurements of P700(+) show that when the cells are depleted of Ci during photosynthesis, P700 becomes more oxidised. This indicates that the resupply of electrons from the intersystem chain is relatively more restricted under conditions of Ci limitation than is the availability of PS 1 electron acceptors. It is proposed that the accumulated Ci pool can directly stimulate the ability of O2 to act as a PS 1 acceptor and that the ability of PS 1 acceptors, such as O2, to relieve restrictions on intersystem electron transfer is perhaps a result of a reduction in cyclic electron flow and a subsequent increase in the oxidation state of the plastoquinone pool.

Electron Flow to the Intersystem Chain from Stromal Components and Cyclic Electron Flow in Maize Chloroplasts, as Detected in Intact Leaves by Monitoring Redox Change of P700 and Chlorophyll Fluorescence

Electron Flow to the Intersystem Chain from Stromal Components and Cyclic Electron Flow in Maize Chloroplasts, as Detected in Intact Leaves by Monitoring Redox Change of P700 and Chlorophyll Fluorescence

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium Synechococcus sp. PCC 7002 as Determined by the Reduction of P700+

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium <italic>Synechococcus</italic> sp. PCC 7002 as Determined by the Reduction of P700⁺

Electron Donation from Cyclic and Respiratory Flows to the Photosynthetic Intersystem Chain is Mediated by Pyridine Nucleotide Dehydrogenase in the Cyanobacterium &lt;italic&gt;Synechocystis&lt;/italic&gt; PCC 6803

Electron Donation from Cyclic and Respiratory Flows to the Photosynthetic Intersystem Chain is Mediated by Pyridine Nucleotide Dehydrogenase in the Cyanobacterium &lt;italic&gt;Synechocystis&lt;/italic&gt; PCC 6803

Pool Size of Electrons That Can Be Donated to P700&lt;sup&gt;+&lt;/sup&gt; As Determined in Intact Leaves: Donation to P700&lt;sup&gt;+&lt;/sup&gt; from Stromal Components Via the Intersystem Chain

Pool Size of Electrons That Can Be Donated to P700&lt;sup&gt;+&lt;/sup&gt; As Determined in Intact Leaves: Donation to P700&lt;sup&gt;+&lt;/sup&gt; from Stromal Components Via the Intersystem Chain

Thylakoid volume, proton translocation and buffering capacity as measured with spin-label techniques

In model studies with lipid vesicles it is shown that the main population of the spin label Tempamine is bound to the membrane surfaces, the piperidine ring directed to the lipid phase. The signal of external label but not of label in the surface is broadened by chromium oxalate. Inner volumes of vesicles can be derived from the partially resolved polar part of the high-field line of Tempone or from the area of the Tempamine spectrum removed by making chromium oxalate enter the vesicles. If this membrane-associated population is corrected for, rotational correlation times for the label in the lumen can be obtained showing that hindrance of rotation is only by a factor of 3–5 instead of 10 as previously reported. In studies with thylakoids volumes of 1–3 μl/mg Chl were found with 0.3 M sorbitol as an osmoticum, 5 mM MgCl 2, 20 mM KCI, and 20 mM chromium oxalate. The internal buffering capacity and the magnitude of pH changes in the inner volume can be determined from flash-induced changes in the amine distribution. The buffering capacity is found to be 7–20 mM in buffer-permeable and approx. 100 mM in buffer-impermeable thylakoids, that is approx. 100 neq per mg Chl. The apparent H +/e −-value in impermeable preparations was found to be up to 0.7 and lower in permeabilized material. ΔpH per flash is 0.04–0.06 units. Possible sources of errors, particularly the presence of non-functional or non-thylakoid membranes, are discussed. Time-resolved signals are presented and several side effects and their suppression are discussed. The response time of the method is up to 2 ms, protons from the donor side of Photosystem II can be separated kinetically from those liberated by the intersystem chain. While transients with less than 2 ms and approx. 20 ms were found with ferricyanide as an electron acceptor in accordance with the results with neutral red, pronounced slow phases ( t 1 2 is several hundred ms) were found without acceptors. Evidence is presented indicating that at least part of these responses do not originate from the thylakoid inner volume.