Dependence of state transitions on illumination time in arabidopsis and barley plants.

Solar energy absorbed by plants can be redistributed between photosystems in the process termed "state transitions" (ST). ST represents a reversible transition of a part of the PSII light harvesting complex (L-LHCII) between photosystem II (PSII) and photosystem I (PSI) in response to the change in light spectral composition. The present work demonstrates a slower development of the state 1 to state 2 transition, i.e., L-LHCII transition from PSII to PSI, in the leaves of dicotyledonous arabidopsis (Arabidopsis thaliana) than in the leaves of monocotyledonous barley (Hordeum vulgare) plants that was assessed by the measurement of chlorophyll a fluorescence at 77 K and of chlorophyll a fluorescence at room temperature. It is known that the first step of the state 1 to state 2 transition is phosphorylation of Lhcb1 and Lhcb2 proteins; however, we detected no difference in the rate of accumulation of these phosphorylated proteins in the studied plants. Therefore, the parameters, which possibly affect the second step of this transition, i.e., the migration of L-LHCII complexes along the thylakoid membrane, were evaluated. Spin-probe EPR measurements demonstrated that the thylakoid membranes viscosity in arabidopsis was higher compared to that in barley. Moreover, confocal microscopy data evidenced the different size of chloroplasts in the leaves of the studied species being larger in arabidopsis. The obtained results suggest that the observed deference in the development of the state 1 to state 2 transition in arabidopsis and barley is caused by the slower L-LHCII migration rate in arabidopsis than in barley plants rather than by the difference in the Lhcb1 and Lhcb2 phosphorylation.

Antioxidants of Non-Enzymatic Nature: Their Function in Higher Plant Cells and the Ways of Boosting Their Biosynthesis.

Plants are exposed to a variety of abiotic and biotic stresses leading to increased formation of reactive oxygen species (ROS) in plant cells. ROS are capable of oxidizing proteins, pigments, lipids, nucleic acids, and other cell molecules, disrupting their functional activity. During the process of evolution, numerous antioxidant systems were formed in plants, including antioxidant enzymes and low molecular weight non-enzymatic antioxidants. Antioxidant systems perform neutralization of ROS and therefore prevent oxidative damage of cell components. In the present review, we focus on the biosynthesis of non-enzymatic antioxidants in higher plants cells such as ascorbic acid (vitamin C), glutathione, flavonoids, isoprenoids, carotenoids, tocopherol (vitamin E), ubiquinone, and plastoquinone. Their functioning and their reactivity with respect to individual ROS will be described. This review is also devoted to the modern genetic engineering methods, which are widely used to change the quantitative and qualitative content of the non-enzymatic antioxidants in cultivated plants. These methods allow various plant lines with given properties to be obtained in a rather short time. The most successful approaches for plant transgenesis and plant genome editing for the enhancement of biosynthesis and the content of these antioxidants are discussed.

Light harvesting regulation: A versatile network of key components operating under various stress conditions in higher plants.

Light harvesting is finetuned through two main strategies controlling energy transfer to the reaction centers of photosystems: i) regulating the amount of light energy at the absorption level, ii) regulating the amount of the absorbed energy at the utilization level. The first strategy is ensured by changes in the cross-section, i.e., the size of the photosynthetic antenna. These changes can occur in a short-term (state transitions) or long-term way (changes in antenna protein biosynthesis) depending on the light conditions. The interrelation of these two ways is still underexplored. Regulating light absorption through the long-term modulation of photosystem II antenna size has been mostly considered as an acclimatory mechanism to light conditions. The present review highlights that this mechanism represents one of the most versatile mechanisms of higher plant acclimation to various conditions including drought, salinity, temperature changes, and even biotic factors. We suggest that H2O2 is the universal signaling agent providing the switch from the short-term to long-term modulation of photosystem II antenna size under these factors. The second strategy of light harvesting is represented by redirecting energy to waste mainly via thermal energy dissipation in the photosystem II antenna in high light through PsbS protein and xanthophyll cycle. In the latter case, H2O2 also plays a considerable role. This circumstance may explain the maintenance of the appropriate level of zeaxanthin not only upon high light but also upon other stress factors. Thus, the review emphasizes the significance of both strategies for ensuring plant sustainability under various environmental conditions.

Cooperative pathway of O2 reduction to H2O2 in chloroplast thylakoid membrane: new insight into the Mehler reaction.

Oxygen reduction in chloroplasts in the light was discovered by (Mehler Arch Biochem Biophys 33:65-77, 1951) as production of hydrogen peroxide. Later, it was shown that the primary product of the oxygen reduction is superoxide radical produced in thylakoids by one-electron transfer from reduced components of photosynthetic electron transport chain to O2 molecule. For a long time, the formation of hydrogen peroxide was considered to be a result of disproportionation of superoxide radicals in chloroplast stroma. Here, we overview a growing number of evidence indicating on another one, additional to disproportionation, pathway of hydrogen peroxide formation in chloroplasts, namely its formation in thylakoid membrane due to reaction of superoxide radical generated in the membrane with the reduced plastoquinone molecule, plastohydroquinone. Since various components of photosynthetic electron transport chain (primarily photosystem I) can supply superoxide radicals to this reaction, we refer this two-step O2 photoreduction to H2O2 as a cooperative process. The significance of hydrogen peroxide production via this pathway for redox signaling and scavenging of reactive oxygen species is discussed.

Inhibition of plastoquinol oxidation at the cytochrome b6f complex by dinitrophenyl ether of iodonitrothymol (DNP-INT) depends on irradiance and H+ uptake by thylakoid membranes.

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.

Alpha Carbonic Anhydrase 5 Mediates Stimulation of ATP Synthesis by Bicarbonate in Isolated Arabidopsis Thylakoids.

We studied bicarbonate-induced stimulation of photophosphorylation in thylakoids isolated from leaves of Arabidopsis thaliana plants. This stimulation was not observed in thylakoids of wild-type in the presence of mafenide, a soluble carbonic anhydrase inhibitor, and was absent in thylakoids of two mutant lines lacking the gene encoding alpha carbonic anhydrase 5 (αCA5). Using mass spectrometry, we revealed the presence of αCA5 in stromal thylakoid membranes of wild-type plants. A possible mechanism of the photophosphorylation stimulation by bicarbonate that involves αCA5 is proposed.

Advances in understanding the physiological role and locations of carbonic anhydrases in C3 plant cells.

The review describes the structures of plant carbonic anhydrases (CAs), enzymes catalyzing the interconversion of inorganic carbon forms and belonging to different families, as well as the interaction of inhibitors and activators of CA activity with the active sites of CAs in representatives of these families. We outline the data that shed light on the location of CAs in green cells of C3 plants, algae and angiosperms, with the emphasis on the recently obtained data. The proven and proposed functions of CAs in these organisms are listed. The possibility of the involvement of several chloroplast CAs in acceleration of the conversion of bicarbonate to CO2 and in supply of CO2 for fixation by Rubisco is particularly considered. Special attention is paid to CAs in various parts of thylakoids and to discussion about current knowledge of their possible physiological roles. The review states that, despite the significant progress in application of the mutants with suppressed CAs synthesis, the approach based on the use of the inhibitors of CA activity in some cases remains quite effective. Combination of these two approaches, namely determining the effect of CA activity inhibitors in plants with certain knocked-out CA genes, turns out to be very useful for understanding the functions of other CAs.

Regulation of the size of photosystem II light harvesting antenna represents a universal mechanism of higher plant acclimation to stress conditions.

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.

Minimizing an Electron Flow to Molecular Oxygen in Photosynthetic Electron Transfer Chain: An Evolutionary View.

Recruitment of H2O as the final donor of electrons for light-governed reactions in photosynthesis has been an utmost breakthrough, bursting the evolution of life and leading to the accumulation of O2 molecules in the atmosphere. O2 molecule has a great potential to accept electrons from the components of the photosynthetic electron transfer chain (PETC) (so-called the Mehler reaction). Here we overview the Mehler reaction mechanisms, specifying the changes in the structure of the PETC of oxygenic phototrophs that probably had occurred as the result of evolutionary pressure to minimize the electron flow to O2. These changes are warranted by the fact that the efficient electron flow to O2 would decrease the quantum yield of photosynthesis. Moreover, the reduction of O2 leads to the formation of reactive oxygen species (ROS), namely, the superoxide anion radical and hydrogen peroxide, which cause oxidative stress to plant cells if they are accumulated at a significant amount. From another side, hydrogen peroxide acts as a signaling molecule. We particularly zoom in into the role of photosystem I (PSI) and the plastoquinone (PQ) pool in the Mehler reaction.

International conference on "Photosynthesis and Hydrogen Energy Research for Sustainability-2019": in honor of Tingyun Kuang, Anthony Larkum, Cesare Marchetti, and Kimiyuki Satoh.

The 10th International Conference on «Photosynthesis and Hydrogen Energy Research for Sustainability-2019¼ was held in honor of Tingyun Kuang (China), Anthony Larkum (Australia), Cesare Marchetti (Italy), and Kimiyuki Satoh (Japan), in St. Petersburg (Russia) during June 23-28, 2019. The official conference organizers from the Russian side were from the Institute of Basic Biological Problems of the Russian Academy of Sciences (IBBP RAS), Russian Society for Photobiology (RSP), and the Komarov Botanical Institute of the Russian Academy of Sciences ([K]BIN RAS). This conference was organized with the help of Monomax Company, a member of the International Congress Convention Association (ICCA), and was supported by the Ministry of Education and Science of the Russian Federation. Here, we provide a brief description of the conference, its scientific program, as well as a brief introduction and key contributions of the four honored scientists. Further, we emphasize the recognition given, at this conference, to several outstanding young researchers, from around the World, for their research in the area of our conference. A special feature of this paper is the inclusion of photographs provided by one of us (Tatsuya Tomo). Lastly, we urge the readers to watch for information on the next 11th conference on "Photosynthesis and Hydrogen Energy Research for Sustainability-2021," to be held in Bulgaria in 2021.

The role of carbonic anhydrase α-CA4 in the adaptive reactions of photosynthetic apparatus: the study with α-CA4 knockout plants.

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.

Carbonic Anhydrases in Photosynthesizing Cells of C3 Higher Plants.

The review presents data on the location, nature, properties, number, and expression of carbonic anhydrase genes in the photosynthesizing cells of C3 plants. The available data about the presence of carbonic anhydrases in plasma membrane, cytoplasm, mitochondria, chloroplast stroma and thylakoids are scrutinized. Special attention was paid to the presence of carbonic anhydrase activities in the different parts of thylakoids, and on collation of sources of these activities with enzymes encoded by the established genes of carbonic anhydrases. The data are presented to show that the consistent incorporation of carbonic anhydrases belonging to different families of these enzymes forms a coherent system of CO2 molecules transport from air to chloroplasts in photosynthesizing cells, where they are included in organic molecules in the carboxylation reaction. It is discussed that the manifestation of the activity of a certain carbonic anhydrase depends on environmental conditions and the stage of ontogenesis.

Antioxidant and signaling functions of the plastoquinone pool in higher plants.

The review covers data representing the plastoquinone pool as the component integrated in plant antioxidant defense and plant signaling. The main goal of the review is to discuss the evidence describing the plastoquinone-involved biochemical reactions, which are incorporated in maintaining the sustainability of higher plants to stress conditions. In this context, the analysis of the reactions of various redox forms of plastoquinone with oxygen species is presented. The review describes how these reactions can constitute both the antioxidant and signaling functions of the pool. Special attention is paid to the reaction of superoxide anion radicals with plastohydroquinone molecules, producing hydrogen peroxide as signal molecules. Attention is also given to the processes affecting the redox state of the plastoquinone pool because the redox state of the pool is of special importance for antioxidant defense and signaling.

Oxidation of the plastoquinone pool in chloroplast thylakoid membranes by superoxide anion radicals.

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 ( O 2 · - ), which may react with the PQ-pool. Here, using isolated thylakoids, we show for the first time the oxidation of the PQ-pool by O 2 · - . The xanthine-xanthine oxidase system was used to supply O 2 · - externally 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  O 2 · - produced in Photosystem I with reduced PQ (plastohydroquinone) creates hydrogen peroxide, which serves as a messenger that signals the redox state of the PETC.

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.

Involvement of the chloroplast plastoquinone pool in the Mehler reaction.

Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler reaction, has been studied using isolated pea thylakoids. The role of the plastoquinone pool in the Mehler reaction was investigated in the presence of dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT), the inhibitor of plastohydroquinone oxidation by cytochrome b6/f complex. Oxygen reduction rate in the presence of DNP-INT was higher than in the absence of the inhibitor in low light at pH 6.5 and 7.6, showing that the capacity of the plastoquinone pool to reduce molecular oxygen in this case exceeded that of the entire electron transfer chain. In the presence of DNP-INT, appearance of superoxide anion radicals outside thylakoid membrane represented approximately 60% of the total superoxide anion radicals produced. The remaining 40% of the produced superoxide anion radicals was suggested to be trapped by plastohydroquinone molecules within thylakoid membrane, leading to the formation of hydrogen peroxide (H2 O2 ). To validate the reaction of superoxide anion radical with plastohydroquinone, xanthine/xanthine oxidase system was integrated with thylakoid membrane in order to generate superoxide anion radical in close vicinity of plastohydroquinone. Addition of xanthine/xanthine oxidase to the thylakoid suspension resulted in a decrease in the reduction level of the plastoquinone pool in the light. The obtained data provide additional clarification of the aspects that the plastoquinone pool is involved in both reduction of oxygen to superoxide anion radicals and reduction of superoxide anion radicals to H2 O2 . Significance of the plastoquinone pool involvement in the Mehler reaction for the acclimation of plants to light conditions is discussed.

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.

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.

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.

Putative function of cytochrome b559 as a plastoquinol oxidase.

The function of cytochrome b559 (cyt b559) in photosystem II (PSII) was studied in a tobacco mutant in which the conserved phenylalanine at position 26 in the beta-subunit was changed to serine. Young leaves of the mutant showed no significant difference in chloroplast ultra structure or in the amount and activity of PSII, while in mature leaves the size of the grana stacks and the amount of PSII were significantly reduced. Mature leaves of the mutant showed a higher susceptibility to photoinhibition and a higher production of singlet oxygen, as shown by spin trapping electron paramagnetic resonance (EPR) spectroscopy. Oxygen consumption and superoxide production were studied in thylakoid membranes in which the Mn cluster was removed to ensure that all the cyt b559 was present in its low potential form. In thylakoid membranes, from wild-type plants, the larger fraction of superoxide production was 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive. This type of superoxide formation was absent in thylakoid membranes from the mutant. The physiological importance of the plastoquinol oxidation by cyt b559 for photosynthesis is discussed.