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The carbonic anhydrase (CA) activities of the preparations of cytoplasm, mitochondria, chloroplast stroma, and chloroplast thylakoids, as well as the expression levels of genes encoding αCA1, αCA2, αCA4, βCA1, βCA2, βCA3, βCA4, βCA5, and βCA6, were measured in the leaves of

The present chapter describes the mechanisms of reactive oxygen species formation in photosynthetic reactions and the functional significance of reactive oxygen species as signal messengers in photosynthetic cells of plants. Attention is given to the acclimation mechanisms of higher plants to abiotic and biotic factors such as increased light, drought, soil salinity and colonization of plants by rhizosphere microorganisms. Special attention is paid to the reactions of reactive oxygen species with the components of the chloroplasts plastoquinone pool leading to production of hydrogen peroxide as a signal molecule, which is involved in acclimation of plants to these stress conditions. The chapter also presents the data demonstrating that regulation of the size of the light-harvesting antenna of photosystem II is one of the universal mechanisms of the structural and functional reorganization of the photosynthetic apparatus of higher plants exposed to the abiotic and biotic factors. These data were obtained for both model Arabidopsis (Arabidopsis thaliana) plants as well as for agricultural barley (Hordeum vulgare) plants. It is hypothesized that hydrogen peroxide, produced with involvement of the plastoquinone pool components, plays the role of a signaling molecule for regulation of the photosystem II antenna size in higher plants when environmental conditions change.

The effect of the colonization of barley plants (Hordeum vulgare L.) by the associative microorganisms Pseudomonas putida BS3701 on the adaptation of the photosynthetic apparatus to high light intensity was studied. A number of differences were found in the functioning and structure of the photosynthetic apparatus of the control plants and plants colonized by P. putida BS3701 grown at 100 µmol quanta/m2 s. The barley plants colonized by P. putida BS3701 were shown to have an increased resistance to high light intensity (1000 µmol quanta/m2 s) when compared to the control plants. It was established that the adaptive decrease in the PSII antenna size was characterized by the same molecular mechanism in both the control plants and plants colonized by P. putida ВS3701. However, the decrease in PSII antenna size was more pronounced in plants colonized by P. putida ВS3701. It can be assumed that this might be the cause of more efficient protection of the photosynthetic apparatus of plants colonized by P. putida BS3701 against photoinhibition under high illumination.

Inhibitory analysis is a useful tool for studying reactions in the photosynthetic apparatus. After introducing by Aachim Trebst in 1978, dinitrophenylether of iodonitrothymol (DNP-INT), a competitive inhibitor of plastoquinol oxidation at the cytochrome (cyt.) b6f complex, has been widely applied to study reactions occurring in the plastoquinone pool and the cyt. b6f complex. Here we examine the inhibitory efficiency of DNP-INT by implementing three approaches to estimate the extent of blockage of electron flow from the plastoquinone pool to photosystem I in isolated thylakoids from spinach (Spinacia oleracea). We confirm that DNP-INT is a potent inhibitor of electron flow to photosystem I and demonstrate that inhibitory action of DNP-INT depends on irradiance and H+ uptake by thylakoid membranes. Based on these findings, we infer that affinity of the quinol-oxidizing site of the cyt. b6f complex to DNP-INT is increased in the light due to hydrogen bonding between DNP-INT molecules and acidic amino acid residue(s), which is (are) protonated in the light.

The characteristics of the formation of the superoxide radical anion ([Formula: see text]) and hydrogen peroxide by xanthine oxidases isolated from microorganisms and from cow's milk were investigated. The increase in pH led to an increase in the rate of xanthine oxidation with oxygen by both xanthine oxidases. The functioning of xanthine oxidase from milk along with the two-electron reduction of O2 to H2O2 carries through the one-electron reduction of O2 to [Formula: see text], and the rate and the fraction of generation of [Formula: see text] increased with increasing pH. Under operation of the microbial xanthine oxidase, the [Formula: see text] radical was not detected in the medium. The results suggest a difference in the operation of active centers of enzyme from different sources.

The role of α-carbonic anhydrase 4 (α-CA4) in photosynthetic machinery functioning in thylakoid membranes was studied, using Arabidopsis thaliana wild type plants (WT) and the plants with knockout of At4g20990 gene encoding α-CA4 (αCA4-mut) grown both in low light (LL, 80 μmol quanta m−2 s−1) or in high light (HL, 400 μmol quanta m−2 s−1). It was found that a content of PsbS protein, one of determinants of non-photochemical quenching of chlorophyll fluorescence, increased in mutants by 30% and 100% compared with WT plants in LL and in HL, respectively. Violaxanthin cycle pigments content and violaxanthin deepoxidase activity in HL were also higher in αCA4-mut than in WT plants. The content of PSII core protein, D1, when adapting to HL, decreased in WT plants and remained unchanged in mutants. This indicates, that the decrease in the content of Lhcb1 and Lhcb2 proteins in HL (Rudenko et al. Protoplasma 55(1):69-78, 2018) in WT plants resulted from decrease of both Photosystem II (PSII) complex content and content of these proteins in this complex, whereas in αCA4-mut plants from the latter process only. The absence of α-CA4 did not affect the rate of electron transport through Photosystem I (PSI) in thylakoids of mutant vs. WT, but led to 50–80% increase in the rate of electron transport from H2O to QA, evidencing the location of α-CA4 close to PSII. The latter difference may raise the question about its causal connection with the difference in the D1 protein content change during adapting to increased illumination in the presence and the absence of α-CA4.

We investigated acclimatory responses of Arabidopsis plants to drought and salinity conditions before the appearance of obvious signs of damage caused by these factors. We detected changes indicating an increase in the reduction level of the chloroplast plastoquinone pool (PQ pool) 5–7 days after introduction of the stress factors. After 10–14 days, a decrease in the size of PSII light harvesting antenna was observed in plants under conditions of drought and salinity. This was confirmed by a decrease in content of PSII antenna proteins and by downregulation of gene expression levels of these proteins under the stress conditions. No changes in values of performance index and maximum quantum yield of PSII were detected. Under drought and salinity, the content of hydrogen peroxide in leaves was higher than in control leaves. Thus, we propose that reduction of the size of PSII antenna represents one of the universal mechanisms of acclimation of higher plants to stress factors and the downsizing already begins to manifest under mild stress conditions. Both the PQ pool reduction state and the hydrogen peroxide content are important factors needed for the observed rearrangement.

The plastoquinone (PQ)-pool in chloroplast thylakoid membranes is a key electron carrier in the photosynthetic electron transport chain (PETC), and its redox state plays an essential role in the control of plant metabolism. Oxygen reduction in thylakoid membranes produces superoxide anion radicals (mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"mml:msubsupmml:miO/mml:mimml:mrowmml:mn2/mml:mn/mml:mrowmml:mrowmml:mo·/mml:momml:mo-/mml:mo/mml:mrow/mml:msubsup/mml:math), which may react with the PQ-pool. Here, using isolated thylakoids, we show for the first time the oxidation of the PQ-pool bymml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"mml:msubsupmml:miO/mml:mimml:mrowmml:mn2/mml:mn/mml:mrowmml:mrowmml:mo·/mml:momml:mo-/mml:mo/mml:mrow/mml:msubsup/mml:math. The xanthine-xanthine oxidase system was used to supplymml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"mml:msubsupmml:miO/mml:mimml:mrowmml:mn2/mml:mn/mml:mrowmml:mrowmml:mo·/mml:momml:mo-/mml:mo/mml:mrow/mml:msubsup/mml:mathexternally to the thylakoid membrane and the redox state of the PQ-pool was monitored by tracking chlorophyll a fluorescence. We propose that, in vivo, the reaction of mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"mml:msubsupmml:miO/mml:mimml:mrowmml:mn2/mml:mn/mml:mrowmml:mrowmml:mo·/mml:momml:mo-/mml:mo/mml:mrow/mml:msubsup/mml:mathproduced in Photosystem I with reduced PQ (plastohydroquinone) creates hydrogen peroxide, which serves as a messenger that signals the redox state of the PETC.

In arabidopsis plants, with an increase in illumination intensity during growth the extent of reduction of the plastoquinone pool in the photosynthetic electron transport chain increased, whereas the effective quantum yield of photosynthesis decreased. After 5 days of growth under high illumination intensity, these parameters in high light returned to values observed in "shade-adapted" plants in low light. During the same period, the size of the antenna decreased, correlating with a decrease in the amounts of proteins of peripheral pigment-protein complexes. It was found that the decrease in the amounts of these proteins occurred due to suppression of transcription of their genes.

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 dye H(2)DCF-DA, which forms the fluorescent molecule DCF in the reaction with hydrogen peroxide, H(2)O(2), was used to study light-induced H(2)O(2) production in isolated intact chloroplasts and in protoplasts of mesophyll cells of Arabidopsis, pea, and maize. A technique to follow the kinetics of light-induced H(2)O(2) production in the photosynthesizing cells using this dye has been developed. Distribution of DCF fluorescence in these cells in the light has been investigated. It was found that for the first minutes of illumination the intensity of DCF fluorescence increases linearly after a small lag both in isolated chloroplasts and in chloroplasts inside protoplast. In protoplasts of Arabidopsis mutant vtc2-2 with disturbed biosynthesis of ascorbate, the rate of increase in DCF fluorescence intensity in chloroplasts was considerably higher than in protoplasts of the wild type plant. Illumination of protoplasts also led to an increase in DCF fluorescence intensity in mitochondria. Intensity of DCF fluorescence in chloroplasts increased much more rapidly than in cytoplasm. The cessation of cytoplasmic movement under illumination lowered the rate of DCF fluorescence intensity increase in chloroplasts and sharply accelerated it in the cytoplasm. It was revealed that in response to switching off the light, the intensity of fluorescence of both DCF and fluorescent dye FDA increases in the cytoplasm in the vicinity of chloroplasts, while it decreases in the chloroplasts; the opposite changes occur in response to switching on the light again. It was established that these phenomena are connected with proton transport from chloroplasts in the light. In the presence of nigericin, which prevents the establishment of transmembrane proton gradients, the level of DCF fluorescence in cytoplasm was higher and increased more rapidly than in the chloroplasts from the very beginning of illumination. These results imply the presence of H(2)O(2) export from chloroplasts to cytoplasm in photosynthesizing cells in the light; the increase in this export falls in the same time interval as does the cessation of cytoplasmic movement.

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.

The effect of ammonium on ATP synthesis, electron transfer, and light-induced uptake of hydrogen ions in pea chloroplasts was studied. It is shown that the dependence of these reactions on ammonium concentration could be due to effects of two different uncoupling processes. The first process is induced by low ammonium concentrations (

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.