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Significance Extracellular production of the reactive oxygen species (ROS) superoxide results from the one-electron reduction of O2. Nearly all major groups of marine microbes produce extracellular superoxide. In this global estimate of marine microbial superoxide production we determine that dark extracellular superoxide production is ultimately a net sink of dissolved oxygen comparable in magnitude to other major terms in the marine oxygen cycle. This abundant source of superoxide to the marine water column provides evidence that extracellular ROS play a significant role in carbon oxidation and the redox cycling of metals in marine environments. Consideration of this significant reductive flux of dissolved oxygen is essential for field, laboratory, and modeling techniques for determining productivity and oxygen utilization in marine systems., The balance between sources and sinks of molecular oxygen in the oceans has greatly impacted the composition of Earth’s atmosphere since the evolution of oxygenic photosynthesis, thereby exerting key influence on Earth’s climate and the redox state of (sub)surface Earth. The canonical source and sink terms of the marine oxygen budget include photosynthesis, respiration, photorespiration, the Mehler reaction, and other smaller terms. However, recent advances in understanding cryptic oxygen cycling, namely the ubiquitous one-electron reduction of O2 to superoxide by microorganisms outside the cell, remains unexplored as a potential player in global oxygen dynamics. Here we show that dark extracellular superoxide production by marine microbes represents a previously unconsidered global oxygen flux and sink comparable in magnitude to other key terms. We estimate that extracellular superoxide production represents a gross oxygen sink comprising about a third of marine gross oxygen production, and a net oxygen sink amounting to 15 to 50% of that. We further demonstrate that this total marine dark extracellular superoxide flux is consistent with concentrations of superoxide in marine environments. These findings underscore prolific marine sources of reactive oxygen species and a complex and dynamic oxygen cycle in which oxygen consumption and corresponding carbon oxidation are not necessarily confined to cell membranes or exclusively related to respiration. This revised model of the marine oxygen cycle will ultimately allow for greater reconciliation among estimates of primary production and respiration and a greater mechanistic understanding of redox cycling in the ocean.

In this study, the effects of excess NH4+-N on the subcellular accumulation of O2 − and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle were investigated using both histochemical and cytochemical methods. Treatments with ≥ 2.00 and ≥ 5.00 mg L−1 NH4+-N for 5 d significantly increased production of O2 − and H2O2, respectively. The activities of plasma membrane-bound NADPH (nicotinamide adenine dinucleotide phosphate) oxidases and antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, dehydroascorbate reductase and glutathione reductase) were also increased correspondingly. This study also provides the first cytochemical evidence of subcellular accumulation of O2 − and H2O2 in the submerged plants. In the leaves of H. verticillata treated with 20.0 mg L−1 NH4+-N, O2 − dependent DAB precipitates were found primarily on the inner side of the plasma membrane, extracellular space and chloroplasts. H2O2-CeCl3 precipitates were mainly localized on the inner side of the plasma membrane and extracellular space of the mesophyll cells. Treatments with the inhibitors of NADPH oxidase (diphenylene iodonium and imidazole) indicate that NH4+-N-induced production of O2 − and H2O2 in H. verticillata leaves may involve plasma membrane-bound NADPH oxidase. Moreover, low-light treatment decreased NH4+-induced O2 − production, suggesting that alterations in the photosynthetic electron transfer chain due to NH4+ toxicity could lead to O2 − production.

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•−) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O2 reduction within PSI was assumed to take place solely in the distal iron–sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O2 photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron–sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron–sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy. Up to a third of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•−), which occurs principally within photosystem I (PSI) via the Mehler reaction. Strikingly, the precise location as well as the evolutionary role of the reaction have long been a matter of debate. For decades, O2 reduction was assumed to take place solely in the distal iron-sulfur clusters of PSI rather than within the two asymmetrical cofactor branches. Here we demonstrate that under high irradiance, O2 photoreduction by PSI takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant, and from the high rates of O2 photoreduction in PSI complexes lacking iron-sulfur clusters and in a mutant PSI, in which the lifetime of this phyllosemiquinone state is extended 100-fold. On these grounds, we suggest that the Mehler reaction serves as a release valve, functioning only when needed, under conditions where both the distal iron-sulfur clusters of PSI and the mobile ferredoxin pool are over reduced.SIGNIFICANCE STATEMENTPhotosynthesis is the process responsible for the oxygenation of the ancient anoxic atmosphere, and the transformation of inorganic carbon to most of the organic matter on Earth. However, it is less commonly appreciated that the appearance of oxygen in the atmosphere led to the unavoidable opposite process in which oxygen is consumed, thereby producing deleterious oxygen radicals such as the superoxide radical. For almost half a decade, the location of the main site of superoxide radical production in chloroplasts has been a matter of debate. We now provide conclusive evidence that it is located in the phylloquinones(s) within photosystem I.

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.

Humic substances (HSs) can influence the growth and composition of freshwater phytoplankton assemblage. Since HSs contain many phenolic and quinonic moieties and cause growth reductions in eco-physiological field experiments, HSs are considered photosystem II herbicides. To test this specific mode of action in vivo and in vitro, respectively, we used intact cells of the green alga Desmodesmus armatus, as well as thylakoids isolated from spinach (Spinacia oleracea) as a model system for the green algal chloroplast. Photosynthetic electron transport was measured as oxygen evolution and variable chlorophyll fluorescence. The in vivo effect of the artificial humic substance HS1500 on algae consisted of no impact on photosynthesis-irradiance curves of intact green algae compared to untreated controls. In contrast, addition of HS1500 to isolated thylakoids resulted in light-induced oxygen consumption (Mehler reaction) as an in vitro effect. Fluorescence induction kinetics of HS-treated thylakoids revealed a large static quenching effect of HS1500, but no inhibitory effect on electron transport. For the case of intact algal cells, we conclude that the highly hydrophilic and rather large molecules of HS1500 are not taken up in effective quantities and, therefore, cannot interfere with photosynthesis. The in vitro tests show that HS1500 has no inhibitory effect on photosystem II but operates as a weak, oxygen-consuming Hill acceptor at photosystem I. Hence, the results indicate that eco-physiological field experiments should focus more strongly on effects of HSs on extracellular features, such as reducing and red-shifting the underwater light field or influencing nutrient availability by cation exchange within the plankton network.

Zostera marina, a widespread seagrass, evolved from a freshwater ancestor of terrestrial monocots and successfully transitioned into a completely submerged seagrass. We found that its oxygen-evolving complex (OEC) was partially inactivated in response to light exposure, as evidenced by both the increment of the relative variable fluorescence at the K-step and the downregulation of the OEC genes and proteins. This photosynthetic regulation was further addressed at both proteome and physiology levels by an in vivo study. The unchanged content of the ΔpH sensor PsbS protein and the non-photochemical quenching induction dynamics, described by a single exponential function, verified the absence of the fast qE component. Contents and activities of chlororespiration, Mehler reaction, malic acid synthesis, and photorespiration key enzymes were not upregulated, suggesting that alternative electron flows remained unactivated. Furthermore, neither significant production of singlet oxygen nor increment of total antioxidative capacity indicated that reactive oxygen species were not produced during light exposure. In summary, these low electron consumptions may allow Z. marina to efficiently use the limited electrons caused by partial OEC photoinactivation to maintain a normal carbon assimilation level.

This study investigates the role of O2 as an electron acceptor alternative to CO2 in photosynthesis of the common marine angiosperm Zostera marina L. Electron transport rates (ETRs) and non-photochemical quenching (NPQ) of Z. marina were measured under saturating irradiance in synthetic seawater containing 2.2 mM DIC and no DIC with different O2 levels (air-equilibrated levels, 3 % of air equilibrium and restored air-equilibrated levels). Lowering O2 did not affect ETR when DIC was provided, while it caused a decrease in ETR and an increase in NPQ in DIC-free media, indicating that O2 acted as an alternative electron acceptor under low DIC. The ETR and NPQ as a function of irradiance were subsequently assessed in synthetic seawater containing (1) 2.2 mM DIC, air-equilibrated O2; (2) saturating CO2, no O2; and (3) no DIC, air-equilibrated O2. These treatments were combined with glycolaldehyde pre-incubation. Glycolaldehyde caused a marked decrease in ETR in DIC-free medium, indicating significant electron flow supported by photorespiration. Combining glycolaldehyde with O2 depletion completely suppressed ETR suggesting the operation of the Mehler reaction, a possibility supported by the photosynthesis-dependent superoxide production. However, no notable effect of suppressing the Mehler reaction on NPQ was observed. It is concluded that during DIC-limiting conditions, such as those frequently occurring in the habitats of Z. marina, captured light energy exceeds what is utilised for the assimilation of available carbon, and photorespiration is a major alternative electron acceptor, while the contribution of the Mehler reaction is minor.

Exogenous abscisic acid application decreased stomatal conductance (Gs), net photosynthetic rate (Pn), photorespiration rate (Pr), carboxylation efficiency (CE), and actual photochemical efficiency (ΦPSII) but increased the Mehler reaction, antioxidant enzyme activities and H 2 O 2 content in ginger leaves. The observed reopening of the stomata triggered by varying concentrations of external CO 2 indicates that increased intercellular CO 2 concentration (caused by decreased photosynthetic dark reaction activity) promotes stomatal closure under ABA content regulation. By comparing the relationships between H 2 O 2 content, stomatal conductance and ABA content under well-watered conditions compared to osmotic drought stress conditions, we concluded that increased H 2 O 2 content caused by exogenous ABA application also promoted stomata closure in ginger leaves. Thus, CO 2 and H 2 O 2 signalling promote more complete stomatal closure under the influence of ABA in ginger leaves.

International audience; The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO2 availability putatively includes enhanced ROS production in the so-called Mehler reaction. Such conditions are thought to encourage O2 to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical (O2·-) and hydrogen peroxide (H2 O2 ). In contrast, here it is shown in Chlamydomonas reinhardtii that CO2 depletion under high light levels lowered cellular H2 O2 production, and that elevated CO2 levels increased H2 O2 production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H2 O2 production under high CO2 availability. High light levels under low CO2 availability induced photoprotective mechanisms called non-photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE-deficient mutant npq4 produced more H2 O2 than wild-type cells under high light levels, although less so under high CO2 availability, whereas it demonstrated equal or greater enzymatic H2 O2 -degrading capacity. The qT-deficient mutant stt7-9 produced the same H2 O2 as wild-type cells under high CO2 availability. Physiological levels of H2 O2 were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO2 availability wild-type cells behaved like stt7-9 cells stuck in state 1.

In this review, we address bioenergetic pathways in the chloroplast of Chlamydomonas reinhardtii, with a focus on photosynthetic electron transfer. The conversion of solar energy into chemical energy by oxygenic photosynthesis, as performed by plants, green algae and cyanobacteria, supports the life on this planet. The production of oxygen (O2) and the assimilation of carbon dioxide (CO2) into organic matter determine, to a large extent, the composition of our atmosphere. Plant photosynthesis is conducted by a series of reactions that occur mainly in the chloroplast, resulting in light-dependent H2O oxidation, NADP+ reduction and ATP formation. NADPH and ATP, produced by linear electron flow (LEF), are required for carbon fixation via the Calvin-Benson-Bassham (CBB) cycle. Besides, photosynthetic electron transfer may operate in a cyclic electron flow (CEF) mode to satisfy the cellular ATP demand. Electrons derived from LEF may also be diverted to various other metabolic pathways, e.g. via ferredoxin (FDX). In addition, photosynthesis evolved to maximize its outcome while minimizing photooxidative stress. In this regard, mechanisms such as non-photochemical quenching (NPQ) and state transitions regulate energy influx at different light availabilities, which feedback to the proton-motive force (pmf) and the redox state of the plastoquinone/plastoquinol (PQ) pool, thereby also regulating LEF and CEF. To overcome possible limitations in electron transfer at the acceptor side of photosystem (PSI), alternative electron transfer pathways evolved, including flavodiiron proteins (FDPs), allowing safe utilization of O2 as alternative electron acceptor, as well as the hydrogenase, which utilizes two electrons and two protons to produce H2. Nevertheless, reactive oxygen species (ROS) may be formed, e.g. via the Mehler reaction at the acceptor side of PSI, which is why photosynthetic electrons are also utilized in detoxification mechanisms to prevent excessive damage. In conclusion, photosynthetic electron transfer is interwoven in a regulatory network that is aimed at adjusting ATP and NADPH production in a way that electron transfer is not harmful to the cell.

Ranunculus glacialis leaves were tested for their plastid terminal oxidase (PTOX) content and electron flow to photorespiration and to alternative acceptors. In shade-leaves, the PTOX and NAD(P)H dehydrogenase (NDH) content were markedly lower than in sun-leaves. Carbon assimilation/light and C(i) response curves were not different in sun- and shade-leaves, but photosynthetic capacity was the highest in sun-leaves. Based on calculation of the apparent specificity factor of ribulose 1*5-bisphosphate carboxylase/oxygenase (Rubisco), the magnitude of alternative electron flow unrelated to carboxylation and oxygenation of Rubisco correlated to the PTOX content in sun-, shade- and growth chamber-leaves. Similarly, fluorescence induction kinetics indicated more complete and more rapid reoxidation of the plastoquinone (PQ) pool in sun- than in shade-leaves. Blocking electron flow to assimilation, photorespiration and the Mehler reaction with appropriate inhibitors showed that sun-leaves were able to maintain higher electron flow and PQ oxidation. The results suggest that PTOX can act as a safety valve in R. glacialis leaves under conditions where incident photon flux density (PFD) exceeds the growth PFD and under conditions where the plastoquinone pool is highly reduced. Such conditions can occur frequently in alpine climates due to rapid light and temperature changes.

The reduction kinetics of the photo-oxidized primary electron donor P700 in photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 were analyzed within the kinetic model, which considers electron transfer (ET) reactions between P700, secondary quinone acceptor A1, iron-sulfur clusters and external electron donor and acceptors - methylviologen (MV), 2,3-dichloro-naphthoquinone (Cl2NQ) and oxygen. PS I complexes containing various quinones in the A1-binding site (phylloquinone PhQ, plastoquinone-9 PQ and Cl2NQ) as well as F X-core complexes, depleted of terminal iron-sulfur F A/F B clusters, were studied. The acceleration of charge recombination in F X-core complexes by PhQ/PQ substitution indicates that backward ET from the iron-sulfur clusters involves quinone in the A1-binding site. The kinetic parameters of ET reactions were obtained by global fitting of the P700+ reduction with the kinetic model. The free energy gap ΔG 0 between F X and F A/F B clusters was estimated as -130 meV. The driving force of ET from A1 to F X was determined as -50 and -220 meV for PhQ in the A and B cofactor branches, respectively. For PQ in A1A-site, this reaction was found to be endergonic (ΔG 0 = +75 meV). The interaction of PS I with external acceptors was quantitatively described in terms of Michaelis-Menten kinetics. The second-order rate constants of ET from F A/F B, F X and Cl2NQ in the A1-site of PS I to external acceptors were estimated. The side production of superoxide radical in the A1-site by oxygen reduction via the Mehler reaction might comprise ≥0.3% of the total electron flow in PS I.

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.

In oxygenic photosynthesis, light produces ATP plus NADPH via linear electron transfer, i.e. the in-series activity of the two photosystems: PSI and PSII. This process, however, is thought not to be sufficient to provide enough ATP per NADPH for carbon assimilation in the Calvin-Benson-Bassham cycle. Thus, it is assumed that additional ATP can be generated by alternative electron pathways. These circuits produce an electrochemical proton gradient without NADPH synthesis, and, although they often represent a small proportion of the linear electron flow, they could have a huge importance in optimizing CO2 assimilation. In Viridiplantae, there is a consensus that alternative electron flow comprises cyclic electron flow around PSI and the water to water cycles. The latter processes include photosynthetic O-2 reduction via the Mehler reaction at PSI, the plastoquinone terminal oxidase downstream of PSII, photorespiration (the oxygenase activity of Rubisco) and the export of reducing equivalents towards the mitochondrial oxidases, through the malate shuttle. In this review, we summarize current knowledge about the role of the water to water cycles in photosynthesis, with a special focus on their occurrence and physiological roles in microalgae.

The mechanisms of action of reactive oxygen species (ROS) in plant and animal cells, in chloroplasts, mitochondria, and other sub-cellular compartments such as the endoplasmic reticulum and nucleus, which result in deleterious effects as well as intra-organelle and metabolically significant signaling reactions, are discussed, as well as physiological effects induced by the signaling functions of ROS. The role of ROS in chloroplast retrograde signaling and modulation of abiotic stress response and pathogen defense is discussed. Attention is focused on mechanisms of ROS production, and particularly on the intra-organelle reactions mediated through reduction of oxygen to superoxide in the mitochondrial cytochrome bc1 and the chloroplast b6f complexes, for which understanding of the mechanistic details is facilitated by the existence of high resolution crystal structures. Attention is directed to the structure-based details that underlie the more than tenfold larger specific rate of superoxide production in the b6f complex compared to that of the mitochondrial bc1 complex. Because plastoquinol oxidation by the cytochrome b6f complex is known to initiate chloroplast state transitions, the effect of superoxide on the intra-membrane chlorophyll protein kinase that is integral to the cytochrome b6f complex, and which underlies the photosynthetic state transitions that regulate the distribution of light energy between the two photosystems, is considered.

The effect of short-term heat stress on photosynthetic electron transport (photosystems I and II) in chloroplasts of Arabidopsis thaliana deficient in digalactosyl diacylglycerol was investigated. PSII electron transport was characterized by chlorophyll fluorescence rise kinetics while the oxidation–reduction reactions of PSI complexes were studied using leaf-absorbance changes. In wild type plants exposed to temperatures above 36 °C, a progressive damping of the fluorescence rise kinetics, and thus of F v / F m and F v / F 0 , revealed the marked decline in the quantum yield of PSII. This temperature-dependent inactivation of water photolysis and light-induced plastoquinone reduction was more pronounced in Arabidopsis mutants, dgd1-2 and dgd1-3 . At light intensities above 600 μmol m −2 s −1 under normal temperatures for growth, Arabidopsis dgd1-3 was also deficient in its capacity to quench the absorbed light energy nonphotochemically (NPQ) if compared to the NPQ efficiencies of the other two genotypes. The measurements of leaf absorbance changes at 820 nm illustrated the temperature-dependent decline in PSI activity. However, the magnitudes of PSI inhibition were only half of the PSII inhibition, regardless of the plants used. These measurements also showed the suppression of cyclic electron flow around PSI which was temperature-dependent. Leaf exposure above 40 °C resulted in the diversion of electron flow through Mehler reaction in which molecular oxygen acts as terminal acceptor. Notably, in the absence of fully operational PSII and cyclic electron transport around PSI, the decay of the leaf absorption at 820 nm after the cessation of far-red illumination illustrated an enhanced charge recombination in PSI complexes.

Gas exchange, chlorophyll a fluorescence and modulated 820 nm reflection were investigated to explore the development of photosynthesis in Jerusalem artichoke (Helianthus tuberosus L.) leaves from initiation to full expansion. During leaf expansion, photosynthetic rate (Pn) increased and reached the maximal level when leaves were fully expanded. The same change pattern was also found in the stomatal conductance and chlorophyll content. Lower Pn could not be ascribed to the higher stomatal resistance in developing leaves, as intercellular CO2 concentration was not significantly lower in these leaves. Lower Pn partly resulted from the lower actual photochemical efficiency of PSII in developing leaves, as more excited energy was dissipated through non-photochemical quenching. The development of primary photochemical reaction and electron transport in the donor side of PSII was completed in the initiating leaves. However, the development of electron transport in the acceptor side of PSII was not accomplished until leaves were fully expanded, indicated by the change in probability that an electron moves further than primary quinone (ψo). PSI activity changed in parallel with ψo suggesting that PSI cooperated well with PSII during leaf expansion. It should be stressed that the development of carbon fixation process was later than primary photochemical reaction but earlier than photosynthetic electron transport during leaf expansion. The later development of photosynthetic electron transport may reduce the production of reactive oxygen species from Mehler reaction, particularly under low carbon fixation.

Diatoms are frequently exposed to high light (HL) levels, which can result in photoinhibition and damage to PSII. Many microalgae can photoreduce oxygen using the Mehler reaction driven by PSI, which could protect PSII. The ability of Nitzschia epithemioides Grunow and Thalassiosira pseudonana Hasle et Heimdal grown at 50 and 300 μmol photons · m-2 · s-1 to photoreduce oxygen was examined by mass spectrometric measurements of 18O2. Both species exhibited significant rates of oxygen photoreduction at saturating light levels, with cells grown in HL exhibiting higher rates. HL-grown T. pseudonana had maximum rates of oxygen photoreduction five times greater than N. epithemoides, with 49% of electrons transported through PSII being used to reduce oxygen. Exposure to excess light (1,000 μmol photons · m-2 · s-1) produced similar decreases in the operating quantum efficiency of PSII (Fq'/Fm') of low light (LL)- and HL-grown N. epithemoides, whereas HL-grown T. pseudonana exhibited much smaller decreases in Fq'/Fm' than LL-grown cells. HL-grown T. pseudonana and N. epithemioides exhibited greater superoxide and hydrogen peroxide production, higher activities (in T. pseudonana) of superoxide dismutase (SOD) and ascorbate peroxidase (APX), and increased expression of three SOD- and one APX-encoding genes after 60 min of excess light compared to LL-grown cells. These responses provide a mechanism that contributes to the photoprotection of PSII against photodamage. © 2010 Phycological Society of America.

Primary leaves of bean (Phaseolus vulgaris L.) seedlings cultivated for 14 days in a growth chamber on complete (control) and phosphate deficient (−P) Knop liquid medium were used for measurements. The −P leaves were smaller and showed an increased specific leaf area (SLA). Their inorganic phosphate (Pi) concentration was considerably lowered. They did not show any significant changes in chlorophyll (Chl) (a + b) concentration and in their net CO2 assimilation rate when it was estimated under the conditions close to those of the seedlings growth. Light response curves of photosynthetic net O2 evolution (P NO2) of the leaves for the irradiation range up to 500 μmol(photon) m−2 s−1 were determined, using the leaf-disc Clark oxygen electrode. The measurements were taken under high CO2 concentration of about 1 % and O2 concentrations of 21 % or lowered to about 3 % at the beginning of measurement. The results obtained at 21 % O2 and the irradiations close to or higher than those used during the seedlings growth revealed the phosphorus stress suppressive effect on the leaf net O2 evolution, however, no such effect was observed at lower irradiations. Other estimated parameters of P NO2 such as: apparent quantum requirement (QRA) and light compensation point (LCP) for the control and −P leaves were similar. However, with a high irradiation and lowered O2 concentration the rate of P NO2 for the −P leaves was markedly higher than that for the control, in relation to both the leaf area and leaf fresh mass. This difference also disappeared at low irradiations, but the estimated reduced QRA values indicate, under those conditions, the increased yield of photosynthetic light reaction, especially in the −P leaves. The presented results confirm the suggestion that during the initial phase of insufficient phosphate feeding the acclimations in the light phase of photosynthesis, both structural and functional appear. They correspond, probably, to the increased energy costs of carbon assimilation under phosphorus stress, e.g. connected with raised difficulties in phosphate uptake and turnover and enhanced photorespiration. Under the experimental conditions especially advantageous for the dark phase of photosynthesis (saturating CO2 and PAR, low O2 concentration), those acclimations may be manifested as an enhancement of photosynthetic net O2 evolution.

Occurrences whereby cnidaria lose their symbiotic dinoflagellate microalgae (Symbiodinium spp.) are increasing in frequency and intensity. These so-called bleaching events are most often related to an increase in water temperature, which is thought to limit certain Symbiodinium phylotypes from effectively dissipating absorbed excitation energy that is otherwise used for photochemistry. Here, we examined photosynthetic characteristics and hydrogen peroxide (H2 O2 ) production, a possible signal involved in bleaching, from two Symbiodinium types (a thermally "tolerant" A1 and "sensitive" B1) representative of cnidaria-Symbiodinium symbioses of reef-building Caribbean corals. Under steady-state growth at 26°C, a higher efficiency of PSII photochemistry, rate of electron turnover, and rate of O2 production were observed for type A1 than for B1. The two types responded very differently to a period of elevated temperature (32°C): type A1 increased light-driven O2 consumption but not the amount of H2 O2 produced; in contrast, type B1 increased the amount of H2 O2 produced without an increase in light-driven O2 consumption. Therefore, our results are consistent with previous suggestions that the thermal tolerance of Symbiodinium is related to adaptive constraints associated with photosynthesis and that sensitive phylotypes are more prone to H2 O2 production. Understanding these adaptive differences in the genus Symbiodinium will be crucial if we are to interpret the response of symbiotic associations, including reef-building corals, to environmental change.

We investigated effects of nitrogen (N) fertilizer and canopy position on the allocation of N to Rubisco and chlorophyll as well as the distribution of absorbed light among thermal energy dissipation, photochemistry, net CO2 assimilation and alternative electron sinks such as the Mehler reaction and photorespiration. The relative reduction state of the primary quinone receptor of photosystem II (QA) was used as a surrogate for photosystem II (PSII) vulnerability to photoinactivation. Measurements were made on needles from the lower, mid and upper canopy of 21-year-old Pinus radiata D. Don trees grown with (N+) and without (N0) added N fertilizer. Rubisco was 45 to 60% higher in needles of N+ trees than in needles of N0 trees at all canopy positions. Chlorophyll was approximately 80% higher in lower- and mid-canopy needles of N+ trees than of N0 trees, but only approximately 20% higher in upper-canopy needles. Physiological differences between N+ and N0 trees were found only in the lower- and mid- canopy positions. Needles of N+ trees dissipated up to 30% less light energy as heat than needles of N0 trees and had correspondingly more reduced QA. Net CO2 assimilation and the proportions of electrons used by alternative electron sinks such as the Mehler reaction and photorespiration were unaffected by N treatment regardless of canopy position. We conclude that the application of N fertilizer mainly affected the biochemistry and light-use physiology in lower- and mid-canopy needles by increasing the amount of chlorophyll and hence the amount of light harvested. This, however, did not improve photochemistry or safe dissipation, but increased PSII vulnerability to photoinactivation, an effect with likely significant consequences during sunflecks or sudden gap formation.

To better understand the adaptation mechanisms of the photosynthetic apparatus of cotton plants to water deficit conditions, the influence of water deficit on photosynthesis, chlorophyll a fluorescence and the activities of antioxidant systems were determined simultaneously in Gossypium hirsutum L. cv. Xinluzao 45 (upland cotton) and Gossypium barbadense L. cv. Xinhai 21 (pima cotton). Water deficit decreased photosynthesis in both cotton species, but did not decrease chlorophyll content or induce any sustained photoinhibition in either cotton species. Water deficit increased ETR/4 − AG, where ETR/4 estimates the linear photosynthetic electron flux and AG is the gross rate of carbon assimilation. The increase in ETR/4 − AG, which represents an increase in photorespiration and alternative electron fluxes, was particularly pronounced in Xinluzao 45. In Xinluzao 45, water deficit increased the activities of antioxidative enzymes, as well as the contents of reactive oxygen species (ROS), which are related to the Mehler reaction. In contrast, moderate water deficit particularly increased non-photochemical quenching (NPQ) in Xinhai 21. Our results suggest that Xinluzao 45 relied on enhanced electron transport such as photorespiration and the Mehler reaction to dissipate excess light energy under mild and moderate water deficit. Xinhai 21 used enhanced photorespiration for light energy utilisation under mild water deficit but, when subjected to moderate water deficit, possessed a high capacity for dissipating excess light energy via heat dissipation.

Effect of drought on the mechanisms of energy dissipation was evaluated in two-month-old Arachis hypogaea cvs. 57–422, 73–30, and GC 8–35. Plants were submitted to three treatments: control (C), mild water stress (S1), and severe water stress (S2). Photosynthetic performance was evaluated as the Hill and Mehler reactions. These activities were correlated with the contents of the low and high potential forms of cytochrome (cyt) b 559, plastoquinone, cyt b 563, and cyt f. Additionally, the patterns of carotenoids and chlorophylls (Chls), as well as the alterations of Chl a fluorescence parameters were studied. Under mild water stress the regulatory mechanism at the antennae level was effective for 57–422 and GC 8–35, while in the cv. 73–30 an overcharge of photosynthetic apparatus occurred. Relative to this cv., under S1 the stability of carotene and the dissipative cycle around photosystem (PS) 2 became an important factor for the effective protection of the PS2 reaction centres. The cyclic electron flow around PS1 was important for energy dissipation under S1 only for the cvs. 57–422 and 73–30.

After incubation at 42 degrees C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (deltandhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of deltandhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42 degrees C for 6 h, the photochemical activity declined more markedly in deltandhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (deltapH), while the consumption of deltapH was highly inhibited in deltandhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO2 assimilation by providing extra deltapH, thus reducing the photooxidative damage.

Two C4 plants, Miscanthus x giganteus and Cyperus longus L., were grown at suboptimal growth temperatures and the relationships between the quantum efficiencies of photosynthetic electron transport through photosystem II (PSII) (PSII operating efficiency; Fq'/Fm') and CO2 assimilation (phiCO2) in leaves were examined. When M. x giganteus was grown at 10 degrees C, the ratio of the PSII operating efficiency to phiCO2 increased relative to that found in leaves grown at 14 and 25 degrees C. Similar increases in the Fq'/Fm': phiCO2 occurred in the leaves of two C. longus ecotypes when the plants were grown at 17 degrees C, compared to 25 degrees C. These elevations of Fq'/Fm': phiCO2 at low growth temperatures were not attributable to the development of anthocyanins, as has been suggested for maize, and were indicative of the operation of an alternative sink to CO2 assimilation for photosynthetic reducing equivalents, possibly oxygen reduction via a Mehler reaction, which would act as a mechanism for protection of PSII from photoinactivation and damage. Furthermore, in M. x giganteus grown at 10 degrees C, further protection of PSII was effected by a 20-fold increase in zeaxanthin content in dark-adapted leaves, which was associated with much higher levels of non-photochemical quenching of excitation energy, compared to that observed in leaves grown at 14 and 25 degrees C. These differences may explain the long growing season and remarkable productivity of this C4 plant in cool climates, even in comparison to other C4 species such as C. longus, which occur naturally in such climates.

Preparations of photosystem II (PSII) from pea (Pisum sativum L.) leaves were used to study the evolution and reduction of molecular oxygen under photoinhibitory conditions. Under these conditions, the photoinduced oxygen uptake did not exceed 10% of the total oxygen-evolving activity in PSII preparations. Both the Hill and the Mehler reactions were found to occur simultaneously under long-term illumination of PSII preparations with high-intensity light in the presence of potassium ferricyanide. During this light treatment in the presence of potassium ferricyanide, the rate of oxygen uptake increased gradually reaching 30% of the oxygen-evolving activity. The photogeneration of superoxide anion radical at increasing light intensities followed a typical light-response curve with a light saturation at 800 W/m2. The results provide evidence that the Mehler reaction is the major source for superoxide and hydrogen peroxide in PSII preparations under photoinhibitory conditions and that the Mehler reaction in PSII proceeds more effectively at high light intensities. The relatively low and sustained rate of oxygen photoreduction in PSII preparations under photoinhibitory conditions substantiates the hypothesis on the involvement of Mehler reaction in cell signaling and regulation.

Superoxide dismutases (SOD) catalyze the disproportionation of the potentially destructive superoxide anion radical (O 2 , a byproduct of aerobic metabolism) to molecular oxygen and hydrogen peroxide: 2O 2 . + 2H → H 2 O 2 + O 2 . Based on metal cofactors, four known metalloforms of SOD enzymes have been identified: they contain either Fe, Mn, Cu and Zn, or Ni. Orthologs of all metalloforms are present in oxygenic photo-autotrophs. The expression of SOD is highly regulated, with specific metalloforms playing an inducible protective role for specific cellular compartments. The various metalloforms of SOD are not distributed equally within either cyanobacteria or eukaryotic algae. Typically, cyanobacteria contain either an NiSOD alone or combinations of Mn and Ni or Fe and Mn metalloforms (CuZn is rare among the cyanobacteria). The bacillariophytes and rhodophytes retain an active MnSOD, whereas the chlorophytes, haptophytes, and embryophytes have either FeSOD or multiple combinations of Fe, Mn, and CuZnSODs. The NiSOD is a relatively novel SOD and has been generally excluded from evolutionary analyses. In both cyanobacteria and chlorophyte algae, the FeSOD metalloform appears to be associated with PSI, where its primary role is most likely to deactivate reactive oxygen produced by the Mehler reaction. The CuZnSOD also appears to be associated with the plastid but is phylogenetically more restricted in its distribution. In eukaryotic algae, SODs are all nuclear encoded and, based on nucleotide sequence, protein structures, and phylogenetic distributions, appear to have unique evolutionary histories arising from the lateral gene transfer of three distinct genes to the nucleus after the endosymbiotic acquisition of mitochondria and plastids. The varied phylogenetic histories and subcellular localizations suggest significantly different selection on these SOD metalloforms after the endosymbiont organelle-to-host gene transfer.

Polyphenol oxidases (PPOs) have been proposed to function in the Mehler reaction, photoreduction of molecular oxygen by PSI [Physiol. Plant.72 (1988) 659]. Under drought stress where photosynthesis is reduced, the Mehler reaction may provide a nondestructive sink for absorbed light energy not used in photochemistry [Plant Physiol. 112 (1996) 265]. If so, water-stressed plants with suppressed PPO are expected to exhibit photooxidative damage and plants with elevated PPO may show increased stress tolerance. However, in experiments examining drought response of tomato plants with different levels of PPO expression, we report more favorable water relations and decreased photoinhibition in transgenic plants with suppressed PPO (SP plants) compared to nontransformed controls (NT plants) and transgenic plants overexpressing PPO (OP plants). In addition, chlorophyll content is higher in water-stressed SP plants compared to NT and OP plants indicating lower photooxidative stress in SP plants. These results suggest that PPO may have a role in the development of plant water stress and potential for photoinhibition and photooxidative damage that may be unrelated to any effects on the Mehler reaction. As a preliminary attempt to understand the role PPO may play in potentiating stress-induced damage, we examined the expression of PPOs during these experiments. Among the seven-member PPO gene family, we found that in response to water stress, two members, PPO B and D, are transcriptionally upregulated in abscission zones of leaf petioles. In addition, PPO B is induced in leaf veins throughout foliar development and in stem vascular tissues, cortical and pith cells. Similarly, tissue printing of stem tissues following drought stress showed increased immunoreactive PPO in vascular tissues, cortical and pith cells of NT and OP plants. Foliar PPO activity of NT plants also increased throughout development in response to water stress. The physiological role(s) of drought-induced PPO activity remains to be determined. © 2004 Elsevier Ireland Ltd. All rights reserved.

The effects of chilling treatment (4 °C) under low irradiance, LI (100 μmol m−2 s−1) and in the dark on subsequent recovery of photosynthesis in chilling-sensitive sweet pepper leaves were investigated by comparing the ratio of quantum yields of photosystem (PS) 2 and CO2 assimilation, ΦPS2/ΦCO2, measured in normal air (21 % O2, NA) and low O2-air (2% O2, LOA), and by analyzing chlorophyll (Chl) a fluorescence parameters. Chilling treatment in the dark had little effect on Fv/Fm and ΦPS2/ΦCO2, but it caused the decrease of net photosynthetic rate (PN) under saturating irradiance after 6-h chilling treatment, indicating that short-term chilling alone did not induce PS2 photoinhibition. Furthermore, photorespiration and Mehler reaction also did not obviously change during subsequent recovery after chilling stress in the dark. During chilling treatment under LI, there were obvious changes in Fv/Fm and ΦPS2/ΦCO2, determined in NA or LOA. Fv/Fm could recover fully in 4 h at 25 °C, and ΦPS2/ΦCO2 increased at the end of the treatment, as determined in both NA and LOA. During subsequent recovery, ΦPS2/ΦCO2 in LOA decreased faster than in NA. Thus the Mehler reaction might play an important role during chilling treatment under LI, and photorespiration was an important process during the subsequent recovery. The recovery of PN under saturating irradiance determined in NA and LOA took about 50 h, implying that there were some factors besides CO2 assimilation limiting the recovery of photosynthesis. From the progress of reduced P700 and the increase of the Mehler reaction during chilling under LI we propose that active oxygen species were the factors inducing PS1 photoinhibition, which prevented the recovery of photosynthesis in optimal conditions because of the slow recovery of the oxidizable P700.

C 4 grasses of the NAD-ME type ( Astrebla lappacea , Eleusine coracana , Eragrostis superba , Leptochloa dubia , Panicum coloratum , Panicum decompositum ) and the NADP-ME type ( Bothriochloa bladhii, Cenchrus ciliaris, Dichanthium sericeum , Panicum antidotale , Paspalum notatum , Pennisetum alopecuroides , Sorghum bicolor ) were used to investigate the role of O 2 as an electron acceptor during C 4 photosynthesis. Mass spectrometric measurements of gross O 2 evolution and uptake were made concurrently with measurements of net CO 2 uptake and chlorophyll fluorescence at different irradiances and leaf temperatures of 30 and 40 ∞ ∞ ∞ C. In all C 4 grasses gross O 2 uptake increased with increasing irradiance at very high CO 2 partial pressures ( p CO 2 ) and was on average 18% of gross O 2 evolution. Gross O 2 uptake at high irradiance and high p CO 2 was on average 3.8 times greater than gross O 2 uptake in the dark. Furthermore, gross O 2 uptake in the light increased with O 2 concentration at both high CO 2 and the compensation point, whereas gross O 2 uptake in the dark was insensitive to O 2 concentration. This suggests that a significant amount of O 2 uptake may be associated with the Mehler reaction, and that the Mehler reaction varies with irradiance and O 2 concentration. O 2 exchange characteristics at high p CO 2 were similar for NAD-ME and NADP-ME species. NAD-ME species had significantly greater O 2 uptake and evolution at the compensation point particularly at low irradiance compared to NADP-ME species, which could be related to different rates of photorespiratory O 2 uptake. There was a good correlation between electron transport rates estimated from chlorophyll fluorescence and gross O 2 evolution at high light and high p CO 2 .

In the course of dehydration, the gas exchange and chlorophyll (Chl) fluorescence were measured under irradiance of 800 μmol m−2 s−1 in detached apple leaves, and the production of active oxygen species (AOS), hydrogen peroxide (H2O2), superoxide (O2 −), hydroxyl radical (−OH), and singlet oxygen (1O2), were determined. Leaf net photosynthetic rate (P N) was limited by stomatal and non-stomatal factors at slight (2–3 h dehydration) and moderate (4–5 h dehydration) water deficiency, respectively. Photoinhibition occurred after 3-h dehydration, which was defined by the decrease of photosystem 2 (PS2) non-cyclic electron transport (P-rate). After 2-h dehydration, an obvious rise in H2O2 production was found as a result of photorespiration rise. If photorespiration was inhibited by sodium bisulfite (NaHSO3), the rate of post-irradiation transient increase in Chl fluorescence (Rfp) was enhanced in parallel with a slight decline in P-rate and with an increase in Mehler reaction. At 3-h dehydration, leaf P-rate decrease could be blocked by glycine (Gly) or methyl viologen (MV) pre-treatment, and MV was more effective than Gly at moderate drought time. AOS (H2O2 and O2 −), prior to photoinhibition produced from photorespiration and Mehler reaction in detached apple leaves at slight water deficiency, were important in dissipating photon energy which was excess to the demand of CO2 assimilation. So photoinhibition could be effectively prevented by the way of AOS production.

Previous studies have shown that exposure of Arabidopsis leaves to high light (HL) causes a systemic acquired acclimation (SAA) response in the vasculature. It has been postulated that C₄-like photosynthesis in the leaf veins triggers this response via the Mehler reaction. To investigate this proposed connection and extend SAA to other plants, we examined the redox state of NADPH, ascorbate (ASA), and glutathione (GSH) pools; levels and histochemical localization of O₂- and H₂O₂ signals; and activities of antioxidant enzymes in the midvein and leaf lamina of rice, when they were subjected to HL and low light. The results showed that (1) high NADPH/NADP(+) was generated by C₄-like photosynthesis under HL in the midvein and (2) SAA was colocally induced by HL, as indicated by the combined signaling network, including the decrease in redox status of ASA and GSH pools, accumulation of H₂O₂ and O₂- signals, and high superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. The high correlations between these occurrences suggest that the enhanced NADPH/NADP(+) in HL-treated midveins might alter redox status of ASA and GSH pools and trigger H₂O₂ and O₂- signals during SAA via the Mehler reaction. These changes in turn upregulate SOD and APX activities in the midvein. In conclusion, SAA may be a common regulatory mechanism for the adaptation of angiosperms to HL. Manipulation of NADPH/NADP(+) levels by C₄-like photosynthesis promotes SAA under HL stress in the midvein.

The relationship between whole chain photosynthetic electron transport and PSII activity was investigated in Porphyra columbina (Montagne) (Rhodophyta), Ulva australis (Areschoug) (Chlorophyta), and Zonaria crenata ( J. Agardh) (Phaeophyta). Mass spectrometric measurements of gross O2 evolution and gross O2 uptake were combined with simultaneous measurement of pulse-modulated chl fluorescence under a range of irradiances and inorganic carbon (Ci) concentrations. At light-limiting irradiance, a good correlation between gross O2 evolution and the electron transport rate (ETR) calculated from chl fluorescence ((Fm′− Fs)/Fm′) was found in the optically thin species (Ulva and Porphyra). The calculated ETR was equivalent to the theoretical electron requirement in these species but overestimated gross O2 evolution in the thicker species Zonaria. In saturating light, especially when Ci availability was low, ETR overestimated gross O2 evolution in all species. Excess electron flow could not be accounted for by an increase in gross O2 uptake; thus neither Mehler-ascorbate-peroxidase reaction nor the photosynthetic carbon oxidation cycle were enhanced at high irradiance or low C i. Alternative explanations for the loss of correlation include cyclic electron flow around PSII that may be engaged under these conditions or nonphotochemical energy quenching within PSII centers. The loss of correlation between ETR and linear photosynthetic electron flow as irradiance increased from limiting to saturating or at low Ci availability and in the case of optically thick thalli limits the application of this technique for measuring photosynthesis in macroalgae.

A photosystem for solar energy conversion, comprised of a culture of green microalgae supplemented with methyl viologen, is proposed. The capture of solar energy is based on the Mehler reaction. The reduction of methyl viologen by the photosynthetic apparatus and its subsequent reoxidation by oxygen produces hydrogen peroxide. This is a rich-energy compound that can be used as a nonpollutant and efficient fuel. Four different species of green microalgae, Chlamydomonas reinhardtii (21gr) C. reinhardtii (CW15), Chlorella fusca, and Monoraphidium braunii, were tested as a possible biocatalyst. Each species presented a different efficiency level in the transformation of energy. Azide was an efficient inhibitor of the hydrogen peroxide scavenging system while maintaining photosynthetic activity of the microalgae, and thus significantly increasing the production of the photosystem. The strain C. reinhardtii (21gr), among the species studied, was the most efficient with an initial production rate of 185 micromol H(2)O(2)/h x mg Chl and reaching a maximum of 42.5 micromol H(2)O(2)/mg Chl when assayed in the presence of azide inhibitor.

To elucidate how excess light energy is dissipated during water deficit, net photosynthesis (P N ), stomatal conductance (g s ), intercellular CO 2 concentration (c;) and Chl a fluorescence were investigated in control and drought-stressed tomato plants (Lycopersicon esculentum). Gross O 2 evolution (E o ) and gross O 2 uptake (U o ) were determined by a mass spectrometric 16 O 2 / 18 O 2 isotope technique. Under drought stress P N , g s , c i and U o decline. While photochemical fluorescence quenching decreases under water deficit, non-photochemical quenching rises. The maximal efficiency of PSII measured in the dark is not affected by drought; however, in the light, E o decreases under water deficit. The ratio P N /E o falls under stress while the ratio U o /E o increases. We conclude that tomato plants follow a double strategy to avoid photodamage under drought stress conditions: (1) a substantial portion of light energy is emitted as heat and PSII activity is downregulated. This results in a decrease in E o as well as P N and U o . Despite reduced charge separation at PSII, the decline of CO 2 assimilation because of lowered stomatal conductance and metabolic changes results in the need of degrading excessive photosynthetic electrons. (2) Oxygen is used as an alternative electron acceptor in photorespiration or Mehler reaction and U o rises relative to E o .

NH4+-grown plants are more sensitive to light stress than NO3−-grown plants, as indicated by reduced growth and intervenal chlorosis of French bean (Phaseolus vulgaris L.). Measuring the time course of Fv/Fm ratios under photoinhibitory light regimes did not reveal any difference in PS II damage between NO3−- and NH4+-grown plants, in spite of some indications of higher energy quenching in NO3−-grown plants. Also, a direct action of NH4+ as an uncoupler at the thylakoid membrane could be excluded. Instead, biochemical analysis revealed enhanced lipid peroxidation and higher activity of scavenging enzymes in NH4+-grown plants indicating that these plants make use of metabolic pathways with stronger radical formation. Evidence for higher rates of photorespiration in NH4+-grown plants came from experiments showing that electron flux and O2 evolution were decreased by SHAM in NH4+-grown plants, and by antimycin A in NO3−-grown plants. Further, the comparison of electron flux and of photoacoustic measurements of O2 evolution suggested that in NH4+-grown plants the Mehler reaction was also increased, at least in the induction phase. However, the major cause of N form-dependent stress sensitivity is assumed to be in the coupling between photosynthesis and respiration, i.e., NO3−-grown plants can utilize the TCA cycle for the generation of C skeletons for amino acid synthesis, thus improving the ATP: reductant balance, whereas NH4+-grown plants have enhanced rates of photorespiration.

Primary productivity in marine waters is widely estimated by the measurements of 14C incorporation, the underwater light climate, and the absorption spectra of phytoplankton. In bio-optical models the quantum efficiency of carbon fixation derived from 14C incorporation rates, the photosynthetically absorbed radiation derived from the underwater light climate, and the phytoplankton absorption spectra are used to calculate time- and depth-integrated primary productivity. Due to the increased sensitivity of commercially available fluorometers, chlorophyll a in vivo fluorescence became a new tool to assess the photosynthetic activity of phytoplankton. Since fluorescence data yield only relative photosynthetic electron transport rates, a direct conversion into absolute carbon fixation rates is not possible. Here, we report a procedure how this problem can be adressed in freshwater phytoplankton. We adapted a marine bio-optical model to the freshwater situation and tested if this model yields realistic results when applied to a hypertrophic freshwater reservoir. Comparison of primary productivity derived from 14C incorporation to primary productivity derived from Chl a fluorescence showed that the conversion of fluorescence data into carbon fixation rates is still an unsolved problem. Absolute electron transport rates calculated from fluorescence data tend to overestimate primary production. We propose that the observed differences are caused mainly by neglecting the package effect of pigments in phytoplankton cells and by non-carbon related electron flow (e.g., nitrogen fixation). On the other hand, the 14C incorporation rates can be artificially influenced by "bottle effects", especially near the water surface, where photoinhibition, photorespiration, and Mehler reaction can play a major role.

The aim of the present study is to detect the monodehydroascorbic acid (MDA) radical in broad bean (Vicia faba L.) leaves which were treated by vacuum-infiltration in Na2SO3 solution and subsequent centrifugation (sulfitetreated leaves). When sulfite-treated leaves were illuminated with white light, the electron spin resonance (ESR) signal of MDA radical was observed. The level of the MDA radical depended on the concentration of sulfite that was used for vacuum-infiltration and on the light intensity of illumination. The formation of the MDA radical in sulfitetreated leaves was inhibited by DCMU or by replacement of air with N2. Glycolaldehyde also inhibited the formation of MDA radical in sulfite-treated leaves. Catalase activity was decreased by the sulfite treatment without affecting significantly the activities of ascorbate peroxidase (AA-POX) and of peroxidase which preferentially oxidizes phenolics (PhOH-POX). From these results, we conclude that the formation of the MDA radical in sulfite-treated leaves is catalyzed by peroxidases using the H2O2 which is generated by photorespiration and the Mehler reaction.

The photosynthetic linear electron transport rate in excess of that used for CO2 reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C4 plant], Amaranthus cruentus L. (NAD-ME-type C4 plant) and Helianthus annuus L. (C3 plant) leaves at different CO2 and O2 concentrations. The electron transport rate (JF) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500 μmol CO2 · mol−1 and 2% O2. Under high light intensities there was a large excess of JF/4 at 10–100% O2 in the C3 plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C4 photosynthesis is limited by supply of atmospheric CO2 to the C4 cycle, the C3 cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO2 fixation in C4 plants was very sensitive to the presence of O2 in the gas phase, rapidly increasing between 0.01 and 0.1% O2, and at 2% O2 it was about two-thirds of that at 21% O2. This shows the importance of the Mehler O2 reduction as an electron sink, compared with photorespiration in C4 plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C4 photosynthesis.

Measurements of the quantum efficiencies of photosynthetic electron transport through photosystem II (φPSII) and CO2 assimilation (φCO2) were made simultaneously on leaves of maize (Zea mays) crops in the United Kingdom during the early growing season, when chilling conditions were experienced. The activities of a range of enzymes involved with scavenging active O2 species and the levels of key antioxidants were also measured. When leaves were exposed to low temperatures during development, the ratio of φPSII/φCO2 was elevated, indicating the operation of an alternative sink to CO2 for photosynthetic reducing equivalents. The activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and superoxide dismutase and the levels of ascorbate and α-tocopherol were also elevated during chilling periods. This supports the hypothesis that the relative flux of photosynthetic reducing equivalents to O2 via the Mehler reaction is higher when leaves develop under chilling conditions. Lipoxygenase activity and lipid peroxidation were also increased during low temperatures, suggesting that lipoxygenase-mediated peroxidation of membrane lipids contributes to the oxidative damage occurring in chill-stressed leaves.

Photoacoustic signals were measured in expanded tobacco leaves, exposed to a controlled atmosphere by being only partly enclosed within the photoacoustic cell. It was aimed to corroborate the conjecture of Reising and Schreiber (Photosynthesis Research 42: 65-73, 1994) that under exceptionally high CO2 levels (ca. 1–5%) the photobaric uptake contribution reflects CO2 uptake induced by light dependent stromal alkalinization. This is shown here by: (1) the shallower damping of the uptake signal vs. the modulation frequency, compared to a normal oxygen evolution signal; (2) the partial inhibition of the uptake signal under 5% CO2 by nigericin; (3) the complete absence of uptake signals under 5% CO2 in a carbonic-anhydrase-deficient mutant, which gave rather a normal oxygen evolution signal. The photoacoustic signals from the wild type and the transgenic tobacco in air could not be distinguished, indicating that the CO2 uptake signal is negligible under this condition. Uptake photobaric signals were also measured in modulated far-red light (ca. 715–750 nm), following addition of white background light (in light limiting intensity). In normal tobacco under 5% CO2, the background light induced an uptake transient, lasting about a minute, then declining to a low steady level. Significantly smaller transients were obtained under normal air, and in the carbonic-anhydrase deficient mutant also under 5% CO2. Extrapolation to zero frequency of the signal damping vs. modulation frequency, in both tobacco genotypes, suggests however similar magnitudes of the uptake transients. On the other hand, no proportional steady-state uptake was observed for the last two cases. Presumably, the steady uptake under 5% CO2 in modulated far-red light reflects CO2 solubilization, while it is an open question whether the transient could be partly contributed also by oxygen photoreduction by PS I (Mehler reaction). It is reasoned that, under conditions of low light, the respiratory activity results in accumulation of CO2 in the photoacoustic cell, which is sufficient to induce an uptake phenomenon, giving a more satisfactory interpretation for the so-called 'low light state' [Cananni and Malkin (1984) Biochim Biophys Acta 766: 525–532].

The flavoenzyme ferredoxin-NADP+ reductase (FNR) is a member of the cellular defense barrier against oxidative damage in Escherichia coli. We evaluated the responses of chloroplast FNR to methyl viologen, a superoxide radical propagator, in wheat (Triticum aestivum L.) plants and chloroplasts. Treatments with the herbicide showed little effect on the levels of FNR protein or transcripts, indicating that expression of this reductase is not up-regulated by oxidants in plants. Viologens and peroxides caused solubilization of active FNR from the thylakoids into the stroma, converting the enzyme from a membrane-bound NADPH producer to a soluble NADPH consumer. This response appeared specific for FNR, since other thylakoid proteins were unaffected by the treatments. The reductase-binding protein was released together with FNR, suggesting that it might be the target of oxidative modification. Stromal accumulation of a functional NADPH reductase in response to oxidative stress is formally analogous to the induction of FNR synthesis observed in E. coli under similar conditions. FNR solubilization may be playing a crucial role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid. The unchecked accumulation of NADPH might otherwise increase the risks of oxidative damage through a rise in the Mehler reaction rates and/or the production of hydroxyl radicals.

Mutation of the sid gene in Festuca pratensis prevents chlorophyll degradation. The senescing leaves retain their chlorophyll complement and stay green. Nevertheless, CO2 assimilation and ribulose-bisphosphate carboxylase/oxygenase content decline in both mutant and wild-type plants. Photosynthesis and chlorophyll a fluorescence measurements were performed in air and at low oxygen to prevent photorespiration. The maximum extractable activity of ribulose 1,5 bisphosphate carboxylase was higher in the senescent mutant leaves than in those of the wild-type control hut Mas much lower than that observed in the mature leaves of either genotype. The activation state of this enzyme was similar in mutant and wild-type lines at equivalent stages of development. Analysis of chlorophyll a fluorescence quenching with varying irradianco showed similar characteristics for mature leaves of the two genotypes. Genotypic variations in photosystem II (I'SII) efficiency were observed only in the senescent leaves. Photochemical quenching and the quantum efficiency of PSII were greater in the senescent mutant leaves than in (he wild type at a given irradiance. The calculated electron flux through PSII was substantially higher in the mutant with a greater proportion of electrons directed to photorespiration. Maximum catalytic activities of ascorbate peroxidase decreased in senescent compared to mature leaves of both genotypes, while glutathione reductase and monodehydroascorbate reductase were unchanged in both cases. Superoxide dismutase activity was approximately doubled and dehydroascorbate reductase activity was three times higher in senescent leaves compared with the mature leaves of both genotypes. In no case was there a difference in enzyme activities between mutant and wild type at equivalent growth stages. The pool of reduced ascorbate was similar in the mature leaves of the two genotypes, whereas it was significantly higher in the senescent leaves of the mutant compared with the wild type. Conversely, the hydrogen peroxide content was significantly higher in the mature leaves of the wild type than in those of the mutant, but in senescent leaves similar values were obtained. In leaves subjected to chilling stress the reduced ascorbate pool was higher in both mature and senescent leaves of the mutant than in their wild-type counterparts. Similarly, the hydrogen peroxide pool was significantly lower in both mature and senescent leaves of the mutant than in the wild type. We conclude that, in spite of deceased CO2 assimilation, the mutant is capable of high rates of electron Slow. The high ascorbate/hydrogen peroxide ratio observed in the mutant, particularly at low temperatures, suggests that the senescent leaves are not subject to enhanced oxidative stress.

Some aspects of hydrogen peroxide (H2O2) production, scavenging and toxicity in Ulva rigida (Chlorophyta) have been investigated. The seaweed produced H2O2 in light and excretion increased exponentially with increasing light up to 800 μmol photons m-2 s-1 where the excretion rate was 330 μmol (kg Chl a)-1s-1. This corresponded to a concentration of 4 μMH2O2 in the experimental medium after 30 min (density of seaweed: 9·2 kg fresh weight m-3). At 1200 μmol photons m-2 s-1, the excretion rate decreased to 190 μmol (kg Chl a)-1 s-1. At 600 μmol photons m-2 s-1, the excretion caused a concentration of 1·8 μM H2O2 in the experimental seawater; after a further 2 h the concentration decreased to a steady level of c. 0·8 μM. The enzymatic degradation of H2O2 is suggested to be primarily through the activity of ascorbate peroxidase (62 mmol H2O2 (kg Chl a)-1 s-1) since no catalase activity was detected. The H2O2 levels in U. rigida are reduced by diffusion through the plasmalemma. Addition of 1 mM H2O2 caused an 8...

In vivo brominating activity by Eucheuma denticulatum was compared with its production of volatile halocarbons. The ability of the alga to brominate phenol red showed good correlation (p< 0.05, r = 1.0) to the production of bromoform (CHBr3) estimated by gas chromatography. The production of tetrabromophenol or bromoform was raised by exposing the algae to mechanical stress (in the form of cutting), increased light or addition of the herbicide DCMU, prior to the measurements. We suggest that the in vivo brominating activity of E. denticulatum can be used as a simple method to study the physiology of this alga. We also suggest that the activity of the brominating peroxidases is dependent on the intracellular concentration of H2O2, possibly produced by the Mehler reaction. Production of singlet oxygen and triplet chlorophyll by photo-oxidative damage may increase halocarbon release. The amount produced and the species of volatile halocarbons released are dependent on the conditions the algae are subjected to prior and during the experiment. Addition of NaCN or NaN3 inhibited the brominating activity with 95 or 75% of control values.

The light-dependent production of hydroxyl radicals (HO*) by thylakoids, chloroplasts and leaves of Spinacia oleracea was investigated using dimethylsulfoxide as HO' trapping agent. Maximum rates of HO' production by thylakoids as indicated by the formation of methane sulfinic acid were observed under aerobic conditions in the absence of added electron acceptors. They were higher than 2 /anol (mgChlh)"1. Saturation of HO* production occurred at the low photon flux density of 100 /rniol m~2 s"1. Trapping of HO* by dimethylsulfoxide suppressed, but did not eliminate light-dependent inactivation of PSI and II suggesting that HO' formation contributed to the photosensitivity of isolated thylakoids. DCMU inhibited HO' formation. Importantly, methylviologen decreased HO* formation in the absence, but stimulated it in the presence of Fe 3+. In intact chloroplasts, HO' formation became appreciable only after KCN had been added to inhibit effective H2O2 scavenging by ascorbate peroxidase. It was stimulated by ferrisulfate, but not by ferricyanide which does not penetrate the chloroplast envelope. Infiltrated spinach leaves behaved similar in principle to intact chloroplasts in regard to HO* formation but HO' production was very slow if detectable at all by the formation of methylsulfini c acid indicating effective radical detoxification. HO' formation is interpreted to be the result of a Fenton-type reaction which produces HO' in chloroplasts from H2O2 and reduced ferredoxin, when O2 is electron acceptor in the Mehler reaction and radical detoxification reactions are inhibited.

To obtain a clearer understanding of the photosensitization process, we have investigated the effect of photosensitization on the photochemical and non-photochemical energy dissipation in green leaves of wheat ( Triticum aestivum L.) and lettuce ( Lactuca sativa L.), pretreated with 5-aminolaevulinic acid (ALA) for 2–24 h in the dark, using chlorophyll (Chl) fluorescence quenching analysis and measurement of the influence on CO 2 uptake and xanthophyll cycle pigments. In response to dark pretreatment, leaves accumulated high levels of protochlorophyllide (PChlide) and non-metabolized ALA. The dark pretreatment had no effect on the intrinsic photochemical efficiency of photosystem II (PS II). Although quantitative differences exist between wheat and lettuce, exposure to actinic light caused significant effects with a similar overall response pattern in both plant species. Changes in excitation energy dissipation were obtained already by a very low photon flux density of 85 μmol photons m −2 s −1 within a few minutes CO 2 uptake was almost completely suppressed in photosensitized leaves, but they were able to build up the ΔpH necessary to drive de-epoxidation of violaxanthin. Fluorescence quenching analysis revealed that a progressive increase in non-radiative energy dissipation (measured as the non-photochemical quenching of Chl fluorescence, q N ) was paralled by a stronger reduction in the primary quinone electron acceptor of PS II (Q A ) with increasing time of ALA pretreatment in the dark. In the early stages of photosensitization, high-energy state quenching mainly contributed to q N . In the later stages, q N was dominated by a photoinhibitory component together with the photodegradation of xanthophyll cycle pigments, in particular antheraxanthin and zeaxanthin. The latter phenomenon appeared to be promoted in response to a highly increased reduction state of Q A . If ALA was simultaneously applied to leaves with 4,6-dioxoheptanoic acid, which acts as a competitive inhibitor of the enzyme ALA dehydratase, photosensitization disappeared when PChlide synthesis was completely inhibited, but was enhanced when inhibition was incomplete. Electron paramagnetic resonance studies using the spin trapping technique revealed a specific ALA-mediated formation of hydroxyl and carbon-centred radicals in response to actinic light exposure of chloroplasts. On the basis of these findings, a possible role of ALA is proposed: ALA enhances the photosensitizing effect(s) triggered by PChlide.

Photosynthetic activity, chloroplast enzymes, and poly-peptides were compared in green and red (ketocarotenoid-containing) cultures of the microalga Haematococcus pluvialis Flotow. Green cultures, grown at 80 μmol pho-tons.m-2. s-1 in an acetate-containing medium, had a mean generation time of 27 h. Ketocarotenoid accumulation was induced by transfer of green cultures to PO4-deficient medium and exposure to 250 μmol photons.m-2. s-1. Under these conditions, there was no increase in cell number, and the cultures turned red. Relative amounts of enzymes and thylakoid polypeptides in red and green cells were ascertained by immunoprobing with standardization on a chlorophyll (Chl) basis. In red cultures, the level of cytochrome f was greatly decreased (< 1% of green cell level), which is expected to greatly impair the linear electron flow from photosystem (PS) II to PS I. Also, the levels of apoproteins in red cells, namely, of CPI, D2, CP47, LHC I, and ribulose-1, 5-bisphosphate carboxylase were reduced to 15, 18, 29, 48, and 80%, respectively, of those in green cells. Only adenosine triphosphate syn-thase exhibited no significant change in the two types of cultures. The respiration rate of red cultures was much higher (100 μmoles O2. mg Chl-1.h-1) than that of green cells (16 μmoles O2. mg Chl-1.h-1). Conversely, net O2 evolution (at Pmax in green cultures was 80 μmoles O2. mg Chl-1.h-1 but was —40 μmoles O2. mg Chl-1.h-1 in red cultures. PS II activity was demonstrated in broken cells of both green and red cultures, showing activity of 40 and 15 μmoles DCPIP-mg Chl-1.h-1 (with DPC as electron donor), respectively. In contrast, PS I activity measured by the Mehler reaction showed that red rather than green cells had a greater activity (64 vs. 46 μmoles O2. mg Chl-1.h-1, respectively). Thus, in spite of the decline of O2 evolution in red cells, the photosystems were still functional. We postulate that the decline of O2, evolution in red cells is largely attributable to an increase in the respiration rate and the impairment of linear electron flow from PS II to PS I and, to some extent, to a decrease in components of the photosystems.