Marina A. Kozuleva

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

O2 reduction by photosystem I involves phylloquinone under steady-state illumination.

O2 reduction was investigated in photosystem I (PS I) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone‐binding site A1. PS I complexes from WT and menB mutant exhibited different dependencies of O2 reduction on light intensity, namely, the values of O2 reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light‐induced O2 reduction by PS I.

Production of superoxide in chloroplast thylakoid membranes ESR study with cyclic hydroxylamines of different lipophilicity.

Accumulation of nitroxide radicals, DCP or TMT , under illumination of a thylakoid suspension containing either hydrophilic, DCP‐H, or lipophilic, TMT‐H, cyclic hydroxylamines that have high rate constants of the reaction with superoxide radicals, was measured using ESR. A slower accumulation of TMT in contrast with DCP accumulation was explained by re‐reduction of TMT by the carriers of the photosynthetic electron transport chain within the membrane. Superoxide dismutase suppressed TMT accumulation to a lesser extent than DCP accumulation. The data are interpreted as evidencing the production of intramembrane superoxide in thylakoids.

Long-term acclimatory response to excess excitation energy: evidence for a role of hydrogen peroxide in the regulation of photosystem II antenna size

Higher plants possess the ability to trigger a long-term acclimatory response to different environmental light conditions through the regulation of the light-harvesting antenna size of photosystem II. The present study provides an insight into the molecular nature of the signal which initiates the high light-mediated response of a reduction in antenna size. Using barley (Hordeum vulgare) plants, it is shown (i) that the light-harvesting antenna size is not reduced in high light with a low hydrogen peroxide content in the leaves; and (ii) that a decrease in the antenna size is observed in low light in the presence of an elevated concentration of hydrogen peroxide in the leaves. In particular, it has been demonstrated that the ability to reduce the antenna size of photosystem II in high light is restricted to photosynthetic apparatus with a reduced level of the plastoquinone pool and with a low hydrogen peroxide content. Conversely, the reduction of antenna size in low light is induced in photosynthetic apparatus possessing elevated hydrogen peroxide even when the reduction level of the plastoquinone pool is low. Hydrogen peroxide affects the relative abundance of the antenna proteins that modulate the antenna size of photosystem II through a down-regulation of the corresponding lhcb mRNA levels. This work shows that hydrogen peroxide contributes to triggering the photosynthetic apparatus response for the reduction of the antenna size of photosystem II by being the molecular signal for the long-term acclimation of plants to high light.

Oxygen Metabolism in Chloroplast

The molecular mechanism of water oxidation to O2 is still unclear, although many structural details are known and some of the details of the charge accumulating cycle are well worked out (reviewed in Barber, 2008; Brudvig, 2008). The water-oxidizing complex, with a Mn4Ca cluster as the active site, is an integral part of the Photosystem II (PSII), one of the main complexes of the photosynthetic electron transport chain (PETC). When the energy of a quantum of light absorbed by a chlorophyll molecule in this photosystem reaches the reaction center, photochemistry occurs leading to charge separation. The electron is used to reduce plastoquinone, while the electron hole is used to oxidize a Mn ion of the cluster and eventually used to oxidize water. Two sequential photochemical turnovers are required to reduce quinone to quinol, while four sequential turnovers are required to oxidize two water molecules forming O2. It is important to note that the water oxidation/oxygen evolution process is the most easily damaged function of the PETC under stress conditions.

Participation of ferredoxin in oxygen reduction by the photosynthetic electron transport chain

Oxygen reduction by isolated pea thylakoids was studied in the presence of ferredoxin (Fd), Fd + NADP, and cytochrome c. At Fd concentrations optimal for NADP reduction, it contributed 30–50% of the reducing equivalents (as deduced by comparing the rates of oxygen reduction and light oxidation of reduced Fd). The oxygen reduction rate in the presence of Fd + NADP was 3–4 times lower than with Fd alone, and comparable to that with cyt c. It is supposed that the process involves a photosystem I component whose reaction with oxygen depends on the rate of electron efflux from the PS I terminal acceptors, and that this component is phylloquinone.

Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines

Applicability of two lipophilic cyclic hydroxylamines (CHAs), CM-H and TMT-H, and two hydrophilic CHAs, CAT1-H and DCP-H, for detection of superoxide anion radical (O2(∙-)) produced by the thylakoid photosynthetic electron transfer chain (PETC) of higher plants under illumination has been studied. ESR spectrometry was applied for detection of the nitroxide radical originating due to CHAs oxidation by O2(∙-). CHAs and corresponding nitroxide radicals were shown to be involved in side reactions with PETC which could cause miscalculation of O2(∙-) production rate. Lipophilic CM-H was oxidized by PETC components, reducing the oxidized donor of Photosystem I, P700(+), while at the same concentration another lipophilic CHA, TMT-H, did not reduce P700(+). The nitroxide radical was able to accept electrons from components of the photosynthetic chain. Electrostatic interaction of stable cation CAT1-H with the membrane surface was suggested. Water-soluble superoxide dismutase (SOD) was added in order to suppress the reaction of CHA with O2(∙-) outside the membrane. SOD almost completely inhibited light-induced accumulation of DCP(∙), nitroxide radical derivative of hydrophilic DCP-H, in contrast to TMT(∙) accumulation. Based on the results showing that change in the thylakoid lumen pH and volume had minor effect on TMT(∙) accumulation, the reaction of TMT-H with O2(∙-) in the lumen was excluded. Addition of TMT-H to thylakoid suspension in the presence of SOD resulted in the increase in light-induced O2 uptake rate, that argued in favor of TMT-H ability to detect O2(∙-) produced within the membrane core. Thus, hydrophilic DCP-H and lipophilic TMT-H were shown to be usable for detection of O2(∙-) produced outside and within thylakoid membranes.

Ferredoxin:NADP(H) oxidoreductase abundance and location influences redox poise and stress tolerance

The abundance and location of ferredoxin:NADP(H) oxidoreductase in the chloroplast influences free radical production, chloroplast redox poise and plant stress perception. In linear photosynthetic electron transport, ferredoxin:NADP(H) oxidoreductase (FNR) transfers electrons from ferredoxin (Fd) to NADP+. Both NADPH and reduced Fd (Fdred) are required for reductive assimilation and light/dark activation/deactivation of enzymes. FNR is therefore a hub, connecting photosynthetic electron transport to chloroplast redox metabolism. A correlation between FNR content and tolerance to oxidative stress is well established, although the precise mechanism remains unclear. We investigated the impact of altered FNR content and localization on electron transport and superoxide radical evolution in isolated thylakoids, and probed resulting changes in redox homeostasis, expression of oxidative stress markers, and tolerance to high light in planta. Our data indicate that the ratio of Fdred to FNR is critical, with either too much or too little FNR potentially leading to increased superoxide production, and perception of oxidative stress at the level of gene transcription. In FNR overexpressing plants, which show more NADP(H) and glutathione pools, improved tolerance to high-light stress indicates that disturbance of chloroplast redox poise and increased free radical generation may help “prime” the plant and induce protective mechanisms. In fnr1 knock-outs, the NADP(H) and glutathione pools are more oxidized relative to the wild type, and the photoprotective effect is absent despite perception of oxidative stress at the level of gene transcription.

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.

The study of oxygen reduction in photosystem I of higher plants using electron donors for this photosystem in intact thylakoids

Oxygen uptake in the light was investigated in suspensions of isolated pea thylakoids upon inhibition of electron transport from photosystem II by diuron and delivery of electrons to photosystem I by means of artificial donors in the presence of ascorbate. The effects of ascorbate and donors on the process of the reduction of O2 molecules by the components of acceptor side of photosystem I was analyzed. It was shown that DCPIP cannot be used as the donor for photosystem I in the study of this process. Apparently, TMPD applied as a donor does not affect immediately the reaction of the O2 reduction, since an increase in its concentration did not lead to an increase in the oxygen uptake rate in the light. In the experiments with TMPD, an increase in light intensity led to an increase in the oxygen uptake rate, and this fact was interpreted as a consequence of the increase in the apparent rate constant of the reaction of the O2 reduction by the components of the acceptor side of photosystem I.