Photosynthetic Oxygen Research Articles

The Interactive Effects of Elevated CO2 and Ammonium Enrichment on the Physiological Performances of Saccharina japonica (Laminariales, Phaeophyta)

Environmental challenges such as ocean acidification and eutrophication influence the physiology of kelp species. We investigated their interactive effects on Saccharina japonica (Laminariales, Phaeophyta) under two pH conditions [Low, 7.50; High (control), 8.10] and three NH 4 + concentrations (Low, 4; Medium, 60; High, 120 μM). The degree of variation of pH values in the culture medium and inhibition rate of photosynthetic oxygen evolution by acetazolamide were affected by pH treatments. Relative growth rates, carbon, nitrogen, and the C:N ratio in tissue samples were influenced by higher concentrations of NH 4 + . Rates of photosynthetic oxygen evolution were enhanced under elevated CO2 or NH 4 + conditions, independently, but these two factors did not show an interactive effect. However, rates of NH 4 + uptake were influenced by the interactive effect of increased CO2 under elevated NH 4 + treatment. Although ocean acidification and eutrophication states had an impact on physiological performance, chlorophyll fluorescence was not affected by those conditions. Our results indicated that the physiological reactions by this alga were influenced to some extent by a rise in the levels of CO2 and NH 4 + . Therefore, we expect that the biomass accumulation of S. japonica may well increase under future scenarios of ocean acidification and eutrophication.

The mechanism of enhanced wastewater nitrogen removal by photo-sequencing batch reactors based on comprehensive analysis of system dynamics within a cycle

To understand the mechanism of enhanced nitrogen removal by photo-sequencing batch reactors (photo-SBRs), which incorporated microalgal photosynthetic oxygenation into the aerobic phases of a conventional cycle, this study performed comprehensive analysis of one-cycle dynamics. Under a low aeration intensity (about 0.02 vvm), a photo-SBR, illuminated with light at 92.27 μ·mol·m−2·s−1, could remove 99.45% COD, 99.93% NH4+-N, 90.39% TN, and 95.17% TP, while the control SBR could only remove 98.36% COD, 83.51% NH4+-N, 78.96% TN, and 97.75% TP, for a synthetic domestic sewage. The specific oxygen production rate (SOPR) of microalgae in the photo-SBR could reach 6.63 fmol O2·cell−1·h−1. One-cycle dynamics shows that the enhanced nitrogen removal by photo-SBRs is related to photosynthetic oxygenation, resulting in strengthened nitrification, instead of direct nutrient uptake by microalgae. A too high light or aeration intensity could deteriorate anoxic conditions and thus adversely affect the removal of TN and TP in photo-SBRs.

Evolution of real municipal wastewater treatment in photobioreactors and microalgae-bacteria consortia using real-time parameters

In the treatment of real municipal wastewater with photo-sequencing batch reactors (PSBR), operating strategies able to achieve high levels of pollutant removal, but reduce the hydraulic retention time (HRT), are imperative for making microalgae-bacteria consortia more competitive than conventional activated sludge systems. In regard to real-time monitoring, on-line probes like Dissolved Oxygen (DO), pH and oxidation–reduction potential (ORP) are cheap and reliable, but their exploitation has been largely overlooked in PSBRs. This paper proposes the use of DO, pH and ORP profiles to reveal the evolution of wastewater treatment in a PSBR treating real municipal wastewater with a mixed consortium of microalgae and bacteria. The PSBR ensured removal efficiency of 87 ± 5% for COD and 98 ± 2% for TKN without external aeration; indeed, photosynthesis was the only driver of the oxygen production. Considering the combined effects of photosynthetic oxygenation and microbial oxygen consumption, some practical information was gathered to understand the complex profiles of the on-line parameters. During dark and light phases, Zero-DO values, DO and pH raises, and their relative peaks were discussed to evaluate correctly the conclusion of the wastewater treatment and therefore to adjust the duration of the PSBR cycle. In particular: (1) two simultaneous “characteristic points”, “Ammonia valley” (pH profile) and “DO breakpoint” (DO profile), detected univocally the complete ammonium removal; (2) the absolute peaks of DO, pH and ORP at maximum irradiance revealed that wastewater treatment was complete and the cycle could be concluded. In this way, these characteristic points were exploited for the optimization of the PSBR cycle, which was concluded after 15–26 h, reducing the HRT by more than 45%.

Phytoplankton photosynthetic rate measurement using tunable pulsed light induced fluorescence kinetics.

The photosynthetic process of phytoplankton is the basis of the material circulation and energy flow of the ecosystem. The rapid and accurate measurement of phytoplankton photosynthesis rate is of great significance to water ecological environment monitoring, marine resource assessment and global climate change prediction. On the basis of "Bio-Optical" model, a photosynthetic rate measurement method based on tunable pulsed light induced fluorescence kinetics was put forward in this paper. The chlorophyll fluorescence was used as the probe of photosynthesis process, and the phytoplankton photosynthetic rate was evaluated by the photosynthetic electron transport rate. Comparative experiment results showed that the photosynthetic electron transport rate measured by fluorescence kinetic method under different conditions of DCMU, culture light and nutrients (nitrogen) were consistent with the photosynthetic oxygen evolution rate measured by oxygen evolution method, and the correlation coefficient R2 were 0.934, 0.957 and 0.955 respectively.

The energy transfer dynamics in C-phycocyanin hexamer from <italic>Spirulina platensis</italic>

Photosynthesis is an ancient and important conversion of solar light to biological energy in photosynthetic organisms. Phycobilisome (PBS) system in cyanobacteria and red algae is one of two major light-harvesting systems of photosynthetic oxygen organisms. PBS is aggregation of water-soluble phycobiliproteins (PBPs) which are distinctively colored group of disk-shaped macromolecular proteins bearing covalently attached open-chain tetrapyrroles known as phycobilins. PBPs are capable to absorb solar light and transfer energy to the chlorophyll of photosynthetic reaction center with a high efficiency. Light energy absorbed by phycobilins is transmitted between the subunits, and then transmitted between different PBPs, finally to the photoreaction center located on the thylakoid membrane. The energy transfer between PBPs is very fast and high efficiency. Because energy can be transferred in different chromophores and there are a large number of chromophores in PBPs, the energy transfer path of PBPs is complex and diverse. In the past 20 years, energy transfer process of PBPs have been analyzed by time-resolved fluorescence spectroscopy because a large shift of the fluorescence peaks can be clearly observed. For example, Zhang et al. estimated the time constant for energy transfer from PC (rod) to APC (core) as 18 ps and 55 ps based on PBPs crystal structures and time-resolved fluorescence spectra of the rod-core complex. However, due to the technical bottleneck of theoretical simulation and spectral experimental detection, the research on the physical mechanism of photosynthetic energy transfer is relatively slow. In recent years, the ultrafast spectrum time resolution has been continuously improved, which provides the conditions for studying the energy transfer of PBPs. In this paper, the mechanism of energy transfer in C-phycocyanin hexamer from Spirulina platensis was studied by ultrafast time-resolved fluorescence spectroscopy. The energy transfer pathway and the related transfer parameters in the C-phycocyanin hexamer were identified by the fitting of the fluorescence decay curve at different detection wavelengths. It was proved that the energy transfer of C-phycocyanin hexamer had four time components from the experiment, which are 6, 22, 280 and 1470 ps, respectively, then the time components were identified and discussed. This study reflects the energy transfer kinetics between different chromophores in C-phycocyanin and provides important experimental data for solving the relationship between the structure and function of PBPs. At the same time, our study has important value for the understanding of the primary process of photosynthesis. In the future, if the resolution of a streak camera can be raised to femtoseconds, it is possible to find more energy transfer paths, and thus it is expected to make a breakthrough in the ultrafast and ultra-efficient energy transfer mechanism of PBPs.

A Closer Look on Spatiotemporal Variations of Dissolved Oxygen in Waste Stabilization Ponds Using Mixed Models

Dissolved oxygen is an essential controlling factor in the performance of facultative and maturation ponds since both take many advantages of algal photosynthetic oxygenation. The rate of this photosynthesis strongly depends on the time during the day and the location in a pond system, whose roles have been overlooked in previous guidelines of pond operation and maintenance (O&M). To elucidate these influences, a linear mixed effect model (LMM) was built on the data collected from three intensive sampling campaigns in a waste stabilization pond in Cuenca, Ecuador. Within two parallel lines of facultative and maturation ponds, nine locations were sampled at two depths in each pond. In general, the output of the mixed model indicated high spatial autocorrelations of data and wide spatiotemporal variations of the oxygen level among and within the ponds. Particularly, different ponds showed different patterns of oxygen dynamics, which were associated with many factors including flow behavior, sludge accumulation, algal distribution, influent fluctuation, and pond function. Moreover, a substantial temporal change in the oxygen level between day and night, from zero to above 20 mg O2·L−1, was observed. Algal photosynthetic activity appeared to be the main reason for these variations in the model, as it was facilitated by intensive solar radiation at high altitude. Since these diurnal and spatial patterns can supply a large amount of useful information on pond performance, insightful recommendations on dissolved oxygen (DO) monitoring and regulations were delivered. More importantly, as a mixed model showed high predictive performance, i.e., high goodness-of-fit (R2 of 0.94), low values of mean absolute error, we recommended this advanced statistical technique as an effective tool for dealing with high autocorrelation of data in pond systems.

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO3−/CO2 as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α‑carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α‑carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO3−/CO2. Addition of exogenous HCO3− or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.

Nutrient removal and community structure of wastewater-borne algal-bacterial consortia grown in raw wastewater with various wavelengths of light

The use of microalgae-bacteria co-culture systems is attractive for wastewater treatment. On the other hand, naturally developed wastewater-borne algal-bacterial consortia growing in real wastewater has rarely been studied and characterized. In this study, wastewater-borne algal-bacterial consortia was grown for photosynthetic oxygen production, nutrient removal, and biodiversity in real wastewater using light-emitting diodes (LEDs) with various wavelengths (blue, green, red, and white light) and intensities (250, 500, 1,000, and 2000 μmol m−2s−1). Among the four wavelengths, green light showed the highest tolerance to photoinhibition but the energy efficiency for oxygen production was the lowest. In addition, illumination with red and white lights at 500 μmol m−2s− onto 500 mg TSS L−1 appeared to be practical and efficient. In PSBRs operation, the illumination of red and white light resulted in removal ratios of 88.3% and 79.0% for TN, 96.8% and 97.0% for TP, 92.4% and 91.9% for TSS, and an algal biomass productivity of 1.03 and 1.01 g L−1 d−1, respectively. The diversity of bacteria was changed by the different wavelength of light, but the dominant species of algae was not changed. These results are expected to provide valuable technical information on algae-based wastewater treatment systems.

Fe isotopes of a 2.4 Ga hematite-rich IF constrain marine redox conditions around the GOE

The hematite- and manganese-rich Hotazel iron formation, Griqualand West basin, South Africa, was deposited at a key moment in time, close to the GOE between 2.4 and 2.3 Ga. It stratigraphically overlies the Ongeluk Formation, comprising thick flood basalts, which in turn interfinger with and cover the Makganyene Formation diamictites, the inferred remnants of the first Paleoproterozoic Snowball Earth interval. No extensive research has been conducted to date on the basal part of the Hotazel Formation due to poor exposure, though it constitutes an important link between a period of large-scale ice cover, extensive volcanism and the onset of atmospheric oxygenation.Here, we present a detailed petrographic, geochemical and Fe isotope study of a roughly 3-metre-long drill-core exposing the Ongeluk to Hotazel contact. Our results show that after the cessation of Ongeluk volcanism, primary precipitation of Fe(III) oxyhydroxides from the photic surface zone of the original basin became the dominant sedimentation mechanism. Negative δ56Fe values (between −0.26 and −0.50‰) in micro-drilled hematite-rich chert indicate that surface water δ56Fe compositions at the time of deposition were depleted. Yet, δ56Fe and bulk-rock Fe/Mn values are still substantially higher (1–2‰) than those reported higher up in the Mn-rich layers of the Hotazel sequence, suggesting that redox potentials were still comparatively limited during the earliest stages of the Hotazel depositional environment.The base of the Hotazel Formation thus forms a transitional interval between precipitation from essentially ferruginous seawater, as recorded in the Ghaap Group BIFs, and from isotopically and chemically highly evolved surface waters, as demonstrated by the Hotazel Mn-rich layers. In the absence of Fe(II) as a strong reducing agent, large volumes of photosynthetic oxygen may have eventually escaped into the atmosphere, leading to the onset of atmospheric oxygenation. Our results thus contradict previous models that place the onset of the GOE before the Hotazel Formation, concurrent with and mechanistically linked to the Makganyene and Ongeluk events. Instead, we show that the Hotazel basal sediments are still pre-GOE, consistent with their circa 2.4 Ga age and the continuation of MIF-S higher in the stratigraphic record.

Hypoxia tolerance in coral-reef triggerfishes (Balistidae)

Despite high rates of photosynthetic oxygen production during the day, the warm waters of coral reefs are susceptible to hypoxia at night due to elevated respiration rates at higher temperatures that also reduce the solubility of oxygen. Hypoxia may be a challenge for coral-reef fish that hide in the reef to avoid predators at night. Triggerfishes (Balistidae) are found in a variety of reef habitats, but they also are known to find refuge in reef crevices and holes at night, which may expose them to hypoxic conditions. The critical oxygen tension (P crit) was determined as the point below which oxygen uptake could not be maintained to support standard metabolic rate (SMR) for five species of triggerfish. The triggerfishes exhibited similar levels of hypoxia tolerance as other coral-reef and coastal marine fishes that encounter low oxygen levels in their environment. Two species, Rhinecanthus rectangulus and R. aculeatus, had the lowest P crit (~ 3.0 kPa O2), comparable to the most hypoxia-tolerant obligate coral-dwelling gobies, while Odonus niger and Sufflamen bursa were moderately tolerant to hypoxia (P crit ~ 4.5 kPa), and Xanthichthys auromarginatus was intermediate (P crit ~ 3.7 kPa). These differences in P crit were not due to differences in oxygen demand, as all the species had a similar SMR once mass differences were taken into account. The results suggest that triggerfish species are adapted for different levels of hypoxia exposure during nocturnal sheltering within the reef.

Chloride Maintains a Protonated Internal Water Network in the Photosynthetic Oxygen Evolving Complex.

In photosystem II (PSII), water oxidation occurs at a Mn4CaO5 cluster and results in production of molecular oxygen. The Mn4CaO5 cluster cycles among five oxidation states, called Sn states. As a result, protons are released at the metal cluster and transferred through a 35 Å hydrogen-bonding network to the lumen. At 283 K, an infrared band at 2830 cm-1 is assigned to an internal solvated hydronium ion via H218O solvent exchange. This result is similar to a previous report at 263 K. Computations on an oxygen evolving complex model predict that chloride can stabilize a hydronium ion on a network of nine water molecules. In this model, a H3O+ stretching mode at 2738 cm-1 is predicted to shift to higher frequency with bromide and to lower frequency with nitrate substitution. The calculated frequencies were compared to S2-minus-S1 reaction-induced Fourier transform infrared spectra acquired from chloride-, bromide-, or nitrate-containing PSII samples, which were active in oxygen evolution. As predicted, the frequency of the 2830 cm-1 band shifted to higher energy with bromide and to lower energy with nitrate substitution. These results support the conclusion that an internal hydronium ion and chloride play a direct role in an internal proton transfer event during the S1-to-S2 transition.

Oxygen Ebullition From Lakes

AbstractThe exchange of oxygen between lakes and the atmosphere is assumed to be driven by diffusion. Here we show that lakes can emit significant amounts of O2 by emerging gas bubbles—a process called ebullition. We found very high proportions of 17 ± 10% O2 (maximum 34%) in emerging gas bubbles in two shallow eutrophic reservoirs. In the studied reservoirs, O2 emission by ebullition was of similar magnitude as diffusive O2 fluxes. By reanalyzing previous studies, we show that the process is ubiquitous and probably quantitatively relevant in many places. We present evidence that O2 in bubbles originates both from photosynthetic oxygen production and hence bubble formation in the oxic water and from stripping by emerging methane bubbles. Ebullition can turn lakes undersaturated in respect to the atmosphere into a net O2 source. Neglecting O2 ebullition leads to an overestimation of lake internal respiration.

The Green Berry Consortia of the Sippewissett Salt Marsh: Millimeter-Sized Aggregates of Diazotrophic Unicellular Cyanobacteria.

Microbial interactions driving key biogeochemical fluxes often occur within multispecies consortia that form spatially heterogeneous microenvironments. Here, we describe the “green berry” consortia of the Sippewissett salt marsh (Falmouth, MA, United States): millimeter-sized aggregates dominated by an uncultured, diazotrophic unicellular cyanobacterium of the order Chroococcales (termed GB-CYN1). We show that GB-CYN1 is closely related to Crocosphaera watsonii (UCYN-B) and “Candidatus Atelocyanobacterium thalassa” (UCYN-A), two groups of unicellular diazotrophic cyanobacteria that play an important role in marine primary production. Other green berry consortium members include pennate diatoms and putative heterotrophic bacteria from the Alphaproteobacteria and Bacteroidetes. Tight coupling was observed between photosynthetic oxygen production and heterotrophic respiration. When illuminated, the green berries became supersaturated with oxygen. From the metagenome, we observed that GB-CYN1 encodes photosystem II genes and thus has the metabolic potential for oxygen production unlike UCYN-A. In darkness, respiratory activity rapidly depleted oxygen creating anoxia within the aggregates. Metagenomic data revealed a suite of nitrogen fixation genes encoded by GB-CYN1, and nitrogenase activity was confirmed at the whole-aggregate level by acetylene reduction assays. Metagenome reads homologous to marker genes for denitrification were observed and suggest that heterotrophic denitrifiers might co-occur in the green berries, although the physiology and activity of facultative anaerobes in these aggregates remains uncharacterized. Nitrogen fixation in the surface ocean was long thought to be driven by filamentous cyanobacterial aggregates, though recent work has demonstrated the importance of unicellular diazotrophic cyanobacteria (UCYN) from the order Chroococcales. The green berries serve as a useful contrast to studies of open ocean UCYN and may provide a tractable model system to investigate microbial dynamics within phytoplankton aggregates, a phenomenon of global importance to the flux of particulate organic carbon and nitrogen in surface waters.

Effect of CO2-induced seawater acidification on growth, photosynthesis and inorganic carbon acquisition of the harmful bloom-forming marine microalga, Karenia mikimotoi.

Karenia mikimotoi is a widespread, toxic and non-calcifying dinoflagellate, which can release and produce ichthyotoxins and hemolytic toxins affecting the food web within the area of its bloom. Shifts in the physiological characteristics of K. mikimotoi due to CO2-induced seawater acidification could alter the occurrence, severity and impacts of harmful algal blooms (HABs). Here, we investigated the effects of elevated pCO2 on the physiology of K. mikimotoi. Using semi-continuous cultures under controlled laboratory conditions, growth, photosynthesis and inorganic carbon acquisition were determined over 4–6 week incubations at ambient (390ppmv) and elevated pCO2 levels (1000 ppmv and 2000 ppmv). pH-drift and inhibitor-experiments suggested that K. mikimotoi was capable of acquiring HCO3-, and that the utilization of HCO3- was predominantly mediated by anion-exchange proteins, but that HCO3- dehydration catalyzed by external carbonic anhydrase (CAext) only played a minor role in K. mikimotoi. Even though down-regulated CO2 concentrating mechanisms (CCMs) and enhanced gross photosynthetic O2 evolution were observed under 1000 ppmv CO2 conditions, the saved energy did not stimulate growth of K. mikimotoi under 1000 ppmv CO2, probably due to the increased dark respiration. However, significantly higher growth and photosynthesis [in terms of photosynthetic oxygen evolution, effective quantum Yield (Yield), photosynthetic efficiency (α), light saturation point (Ek) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity] were observed under 2000 ppmv CO2 conditions. Furthermore, elevated pCO2 increased the photo-inhibition rate of photosystem II (β) and non-photochemical quenching (NPQ) at high light. We suggest that the energy saved through the down-regulation of CCMs might lead to the additional light stress and photo-damage. Therefore, the response of this species to elevated CO2 conditions will be determined by more than regulation and efficiency of CCMs.

The short-term effects of elevated CO2 and ammonium concentrations on physiological responses in Gracilariopsis lemaneiformis (Rhodophyta)

Ocean acidification (OA) and coastal eutrophication affect coastal marine organisms. We studied the physiological responses of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta) to increased concentrations of CO2 and NH4+. Incubation treatments were applied at two different pH units (low, 7.5; high (control), 7.9) and three different NH4+ concentrations (low, 10; medium, 50; high, 100 μM). Growth, rates of photosynthetic oxygen evolution, and NH4+ uptake rates were affected by both elevated CO2 and NH4+ conditions. The changes in the pH of culture media were influenced by elevated CO2 or NH4+ treatments. However, chlorophyll fluorescence was affected only by the level of NH4+. These results indicate that the physiological responses of G. lemaneiformis might be enhanced when the concentrations of CO2 and NH4+ rise. Therefore, cultures of this alga could provide a good mitigation solution against ongoing problems with OA and coastal eutrophication.

Photosynthetic oxygen production in a warmer ocean: the Sargasso Sea as a case study.

Photosynthetic O2 production can be an important source of oxygen in sub-surface ocean waters especially in permanently stratified oligotrophic regions of the ocean where O2 produced in deep chlorophyll maxima (DCM) is not likely to be outgassed. Today, permanently stratified regions extend across approximately 40% of the global ocean and their extent is expected to increase in a warmer ocean. Thus, predicting future ocean oxygen conditions requires a better understanding of the potential response of photosynthetic oxygen production to a warmer ocean. Based on our own and published observations of water column processes in oligotrophic regions, we develop a one-dimensional water column model describing photosynthetic oxygen production in the Sargasso Sea to quantify the importance of photosynthesis for the downward flux of O2 and examine how it may be influenced in a warmer ocean. Photosynthesis is driven in the model by vertical mixing of nutrients (including eddy-induced mixing) and diazotrophy and is found to substantially increase the downward O2 flux relative to physical-chemical processes alone. Warming (2°C) surface waters does not significantly change oxygen production at the DCM. Nor does a 15% increase in re-mineralization rate (assuming Q10 = 2; 2°C warming) have significant effect on net sub-surface oxygen accumulation. However, changes in the relative production of particulate (POM) and dissolved organic material (DOM) generate relatively large changes in net sub-surface oxygen production. As POM/DOM production is a function of plankton community composition, this implies plankton biodiversity and food web structure may be important factors influencing O2 production in a warmer ocean.This article is part of the themed issue 'Ocean ventilation and deoxygenation in a warming world'.

Tracking Reactive Water and Hydrogen-Bonding Networks in Photosynthetic Oxygen Evolution.

In oxygenic photosynthesis, photosystem II (PSII) converts water to molecular oxygen through four photodriven oxidation events at a Mn4CaO5 cluster. A tyrosine, YZ (Y161 in the D1 polypeptide), transfers oxidizing equivalents from an oxidized, primary chlorophyll donor to the metal center. Calcium or its analogue, strontium, is required for activity. The Mn4CaO5 cluster and YZ are predicted to be hydrogen bonded in a water-containing network, which involves amide carbonyl groups, amino acid side chains, and water. This hydrogen-bonded network includes amino acid residues in intrinsic and extrinsic subunits. One of the extrinsic subunits, PsbO, is intrinsically disordered. This extensive (35 Å) network may be essential in facilitating proton release from substrate water. While it is known that some proteins employ internal water molecules to catalyze reactions, there are relatively few methods that can be used to study the role of water. In this Account, we review spectroscopic evidence from our group supporting the conclusion that the PSII hydrogen-bonding network is dynamic and that water in the network plays a direct role in catalysis. Two approaches, transient electron paramagnetic resonance (EPR) and reaction-induced FT-IR (RIFT-IR) spectroscopies, were used. The EPR experiments focused on the decay kinetics of YZ• via recombination at 190 K and the solvent isotope, pH, and calcium dependence of these kinetics. The RIFT-IR experiments focused on shifts in amide carbonyl frequencies, induced by photo-oxidation of the metal cluster, and on the isotope-based assignment of bands to internal, small protonated water clusters at 190, 263, and 283 K. To conduct these experiments, PSII was prepared in selected steps along the catalytic pathway, the Sn state cycle (n = 0-4). This cycle ultimately generates oxygen. In the EPR studies, S-state dependent changes were observed in the YZ• lifetime and in its solvent isotope effect. The YZ• lifetime depended on the presence of calcium at pH 7.5, but not at pH 6.0, suggesting a two-donor model for PCET. At pH 6.0 or 7.5, barium and ammonia both slowed the rate of YZ• recombination, consistent with disruption of the hydrogen-bonding network. In the RIFT-IR studies of the S state transitions, infrared bands associated with the transient protonation and deprotonation of internal waters were identified by D2O and H218O labeling. The infrared bands of these protonated water clusters, Wn+ (or nH2O(H3O)+, n = 5-6), exhibited flash dependence and were produced during the S1 to S2 and S3 to S0 transitions. Calcium dependence was observed at pH 7.5, but not at pH 6.0. S-state induced shifts were observed in amide C═O frequencies during the S1 to S2 transition and attributed to alterations in hydrogen bonding, based on ammonia sensitivity. In addition, isotope editing of the extrinsic subunit, PsbO, established that amide vibrational bands of this lumenal subunit respond to the S state transitions and that PsbO is a structural template for the reaction center. Taken together, these spectroscopic results support the hypothesis that proton transfer networks, extending from YZ to PsbO, play a functional and dynamic role in photosynthetic oxygen evolution.

Root Bending Is Antagonistically Affected by Hypoxia and ERF-Mediated Transcription via Auxin Signaling.

When plants encounter soil water logging or flooding, roots are the first organs to be confronted with reduced gas diffusion resulting in limited oxygen supply. Since roots do not generate photosynthetic oxygen, they are rapidly faced with oxygen shortage rendering roots particularly prone to damage. While metabolic adaptations to low oxygen conditions, which ensure basic energy supply, have been well characterized, adaptation of root growth and development have received less attention. In this study, we show that hypoxic conditions cause the primary root to grow sidewise in a low oxygen environment, possibly to escape soil patches with reduced oxygen availability. This growth behavior is reversible in that gravitropic growth resumes when seedlings are returned to normoxic conditions. Hypoxic root bending is inhibited by the group VII ethylene response factor (ERFVII) RAP2.12, as rap2.12-1 seedlings show exaggerated primary root bending. Furthermore, overexpression of the ERFVII member HRE2 inhibits root bending, suggesting that primary root growth direction at hypoxic conditions is antagonistically regulated by hypoxia and hypoxia-activated ERFVIIs. Root bending is preceded by the establishment of an auxin gradient across the root tip as quantified with DII-VENUS and is synergistically enhanced by hypoxia and the auxin transport inhibitor naphthylphthalamic acid. The protein abundance of the auxin efflux carrier PIN2 is reduced at hypoxic conditions, a response that is suppressed by RAP2.12 overexpression, suggesting antagonistic control of auxin flux by hypoxia and ERFVII. Taken together, we show that hypoxia triggers an escape response of the primary root that is controlled by ERFVII activity and mediated by auxin signaling in the root tip.

Co-ordination of NDH and Cup proteins in CO2 uptake in cyanobacterium Synechocystis sp. PCC 6803.

High and low affinity CO2-uptake systems containing CupA (NDH-1MS) and CupB (NDH-1MS'), respectively, have been identified in Synechocystis sp. PCC 6803, but it is yet unknown how the complexes function in CO2 uptake. In this work, we found that deletion of cupB significantly lowered the growth of cells, and deletion of both cupA and cupB seriously suppressed the growth below pH 7.0 even under 3% CO2. The rate of photosynthetic oxygen evolution was decreased slightly by deletion of cupA but significantly by deletion of cupB and more severely by deletion of both cupA and cupB, especially in response to changed pH conditions under 3% CO2. Furthermore, we found that assembly of CupB into NDH-1MS' was dependent on NdhD4 and NdhF4. NDH-1MS' was not affected in the NDH-1MS-degradation mutant and NDH-1MS was not affected in the NDH-1MS'-degradation mutants, indicating the existence of independent CO2-uptake systems under high CO2 conditions. The light-induced proton gradient across thylakoid membranes was significantly inhibited in ndhD-deletion mutants, suggesting that NdhDs functions in proton pumping. The carbonic anhydrase activity was suppressed partly in the cupA- or cupB-deletion mutant but severely in the mutant with both cupA and cupB deletion, indicating that CupA and CupB function in conversion of CO2 to HCO3-. In turn, deletion of cup genes lowered the transthylakoid membrane proton gradient and deletion of ndhDs decreased the CO2 hydration. Our results suggest that NDH-1M provides an alkaline region to activate Cup proteins involved in CO2 uptake.

Calcium, Ammonia, Redox-Active Tyrosine YZ, and Proton-Coupled Electron Transfer in the Photosynthetic Oxygen-Evolving Complex.

A redox-active tyrosine, YZ (Y161 in the D1 polypeptide), is essential in photosystem II (PSII), which conducts photosynthetic oxygen evolution. On each step of the light-driven oxygen evolving reaction, YZ radical is formed by a chlorophyll cation radical. YZ radical is then reduced by a Mn4CaO5 cluster in a proton coupled electron transfer (PCET) reaction. YZ is hydrogen bonded to His190-D1 and to water molecules in a hydrogen-bonding network, involving calcium. This network is sensitive to disruption with ammonia and to removal and replacement of calcium. Only strontium supports activity. Here, we use electron paramagnetic resonance (EPR) spectroscopy to define the influence of ammonia treatment, calcium removal, and strontium/barium substitution on YZ radical PCET at two pH values. A defined oxidation state of the metal cluster (S2) was trapped by illumination at 190 K. The net reduction and protonation of YZ radical via PCET were monitored by EPR transients collected after a 532 nm laser flash. At 190 K, YZ radical cannot oxidize the Mn4CaO5 cluster and decays on the seconds time scale by recombination with QA-. The overall decay half-time and biexponential fits were used to analyze the results. The reaction rate was independent of pH in control, calcium-reconstituted PSII (Ca-PSII). At pH 7.5, the YZ radical decay rate decreased in calcium-depleted (CD-PSII) and barium/strontium-reconstituted PSII (Ba-PSII, Sr-PSII), relative to Ca-PSII. At pH 6.0, the YZ radical decay rate was not significantly altered in CD-PSII and Sr-PSII but decreased in Ba-PSII. A two-pathway model, involving two competing proton donors with different pKa values, is proposed to explain these results. Ammonia treatment decreased the YZ decay rate in Ca-PSII, Sr-PSII, and CD-PSII, consistent with a reaction that is mediated by the hydrogen-bonding network. However, ammonia treatment did not alter the rate in Ba-PSII. This result is interpreted in terms of the large ionic radius of barium and the elevated pKa of barium-bound water, which are expected to disrupt hydrogen bonding. In addition, evidence for a functional interaction between the S2 protonated water cluster (Wn+) and the YZ proton donation pathway is presented. This interaction is proposed to increase the rate of the YZ PCET reaction.