B. N. Ivanov

Production and diffusion of chloroplastic H2O2 and its implication to signalling

Hydrogen peroxide (H(2)O(2)) is recognized as an important signalling molecule. There are two important aspects to this function: H(2)O(2) production and its diffusion to its sites of action. The production of H(2)O(2) by photosynthetic electron transport and its ability to diffuse through the chloroplast envelope membranes has been investigated using spin trapping electron paramagnetic resonance spectroscopy and H(2)O(2)-sensitive fluorescence dyes. It was found that, even at low light intensity, a portion of H(2)O(2) produced inside the chloroplasts can leave the chloroplasts thus escaping the effective antioxidant systems located inside the chloroplast. The production of H(2)O(2) by chloroplasts and the appearance of H(2)O(2) outside chloroplasts increased with increasing light intensity and time of illumination. The amount of H(2)O(2) that can be detected outside the chloroplasts has been shown to be up to 5% of the total H(2)O(2) produced inside the chloroplasts at high light intensities. The fact that H(2)O(2) produced by chloroplasts can be detected outside these organelles is an important finding in terms of understanding how chloroplastic H(2)O(2) can serve as a signal molecule.

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.

The production and scavenging of reactive oxygen species in the plastoquinone pool of chloroplast thylakoid membranes

Reactive oxygen species (ROS) resulting from oxygen reduction, superoxide anion radical O2(*-) and hydrogen peroxide H(2)O(2) are very significant in the cell metabolism of aerobic organisms. They can be destructive and lead to apoptosis and they can also serve as signal molecules. In the light, chloroplasts are known to be one of the main sources of ROS in plants. However, the components involved in oxygen reduction and the detailed chemical mechanism are not yet well established. The present review describes the experimental data and theoretical considerations that implicate the plastoquinone pool (PQ-pool) in this process. The evidence indicates that the PQ-pool has a dual role: (1) the reduction of O(2) by plastosemiquinone to superoxide and (2) the reduction of superoxide by plastohydroquinone to hydrogen peroxide. The second role represents not only the scavenging of superoxide, but also the generation of hydrogen peroxide as an important signaling molecule. The regulatory and protective functions of the PQ-pool are discussed in the context of these reactions.

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.

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H+ (mg Chl)−1 h−1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal β-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO3− dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H+ (mg Chl)−1 h−1) were observed in the presence of detergents (Triton X-100 or n-octyl-β-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H+ (mg Chl)−1 h−1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.

Light-induced stimulation of carbonic anhydrase activity in pea thylakoids

Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6–8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light‐generated proton gradient across the thylakoid membrane thus facilitating the production of CO2 from HCO3 − and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.

Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: evidence with a specific fluorescent probe.

The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2). These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ > 600 nm; 1500 μmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.

Diversity in forms and functions of carbonic anhydrase in terrestrial higher plants

The review summarizes current data on the existence in terrestrial higher plants of several carbonic anhydrase forms differing in their properties, molecular structure, and intracellular localization. Possible functions of these carbonic anhydrases are discussed as well as specific features of carbon-concentrating mechanisms in phototrophic tissues of plants with C3 and C4 pathways of photosynthesis.

Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex.

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 μmol O2/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl2 or diphenylcarbazide, or diuron as an inhibitor of electron transfer from QA, the primary bound quinone acceptor, to QB, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to Fmax, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680+ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H2O2, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O2 with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680+, as well as by oxygen reduction by still unidentified components of PSII.

Photosystem II associated carbonic anhydrase activity in higher plants is situated in core complex

The thylakoid membrane containing photosystem II (PSII membranes) from pea and wheat leaves catalyzed the reaction of CO2 hydration with low rate, which increased after their incubation either with Triton X‐100, up to Triton/chlorophyll ratio 1:1, or 1 M CaCl2. The presence of the inhibitor of CAs, p‐aminomethylbenzensulfonamide (mafenide), at the start line in the course of electrophoresis of PSII membranes solubilized by n‐dodecyl‐β‐maltoside (DM) decreased the amount of PSII core complex in the gel. The elution of PSII core complex from the column with immobilized mafenide occurred only either by mafenide or another inhibitor of CAs, ethoxyzolamide. The above results led to a conclusion that membrane‐bound CA activity associated with PSII is situated in the core complex.