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In a rapidly warming world, we ask, "What limits the potential of marine diatoms to acclimate to elevated temperatures?," a group of ecologically successful unicellular eukaryotic photoautotrophs that evolved in a cooler ocean and are critical to marine food webs. To this end, we examined thermal tolerance mechanisms related to photosynthesis in the sequenced and transformable model diatom Phaeodactylum tricornutum. Data from transmission electron microscopy (TEM) and fatty acid methyl ester-gas chromatography mass spectrometry (FAME-GCMS) suggest that saturating thylakoid-associated fatty acids allowed rapid (on the order of hours) thermal tolerance up to 28.5°C. Beyond this critical temperature, thylakoid ultrastructure became severely perturbed. Biophysical analyses revealed that electrochemical leakage through the thylakoid membranes was extremely sensitive to elevated temperature (Q10 of 3.5). Data suggest that the loss of the proton motive force (pmf) occurred even when heat-labile photosystem II (PSII) was functioning, and saturation of thylakoid-associated fatty acids was active. Indeed, growth was inhibited when leakage of pmf through thylakoid membranes was insufficiently compensated by proton input from PSII. Our findings provide a mechanistic understanding of the importance of rapid saturation of thylakoid-associated fatty acids for ultrastructure maintenance and a generation of pmf at elevated temperatures. To the extent these experimental results apply, the ability of diatoms to generate a pmf may be a sensitive parameter for thermal sensitivity diagnosis in phytoplankton.

Phytoplankton are extraordinarily diverse, comprising 13 phylogenetic groups, with diatoms, dinoflagellates, and haptophytes among the most prominent eukaryotes in the ocean. Development of sensor technologies for rapid taxonomic and physiological analysis of phytoplankton communities is crucial for ecological monitoring programs in the global ocean. We describe a novel, ultra-sensitive, multi-spectral fluorescence induction and relaxation instrument (a mini-FIRe) and examine its analytical capability of rapidly determining phytoplankton taxonomic groups, as well as physiological characteristics and photosynthetic rates. We collected and analyzed the database of spectral and photosynthetic properties of major taxonomic groups of phytoplankton. We revealed that the spectral shape of the functional absorption cross-section of Photosystem II (PSII), sPSII(lex), is remarkably constrained within each major phylogenetic group of eukaryotic phytoplankton, including diatoms, haptophytes, dinoflagellates, and chlorophytes. Variability in sPSII(lex) within each group was significantly smaller than the difference between groups. We also examined the classical excitation spectra of chl a fluorescence yields, Fm(lex). Our comparative analysis revealed that sPSII(lex) is a better and more specific proxy for taxonomic analysis. For instance, our developed sPSII-based algorithm correctly identified 90% of experimental data, compared to 77% identified by the Fm-based algorithm. Our results suggest that the multi-color variable fluorescence analysis offers a tool for combined physiological and taxonomic analysis, including identification of major phyla within the ‘red’ lineage of eukaryotic phytoplankton.

Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5-1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor-acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor-acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.

Biological hotspots along the West Antarctic Peninsula (WAP) are characterized by high phytoplankton productivity and biomass as well as spatially focused penguin foraging activity. While unique physical concentrating processes were identified in one of these hotspots, understanding the mechanisms driving the blooms at these locations is of high importance. Factors posited to explain the blooms include the upwelling of macronutrient‐ and micronutrient‐enriched modified Upper Circumpolar Deep Water (mUCDW) and the depth of the mixed layer influencing overall light availability for phytoplankton. Using shipboard trace‐metal clean incubation experiments in three different coastal biological hotspots spanning a north‐south gradient along the WAP, we tested the Canyon Hypothesis (upwelling) for enhanced phytoplankton growth. Diatoms dominated the Southern region, while the Northern region was characterized by a combination of diatoms and cryptophytes. There was ample concentration of macronutrients at the surface and no phytoplankton growth response was detected with the addition of nutrient‐enriched mUCDW water or iron solution to surface waters. For all treatments, addition of mUCDW showed no enhancement in phytoplankton growth, suggesting that local upwelling of nutrient‐enriched deep water in these hotspots was not the main driver of high phytoplankton biomass. Furthermore, the dynamics in the photoprotective pigments were consistent with the light levels used during these incubations showing that phytoplankton are able to photoacclimate rapidly to higher irradiances and that in situ cells are low light adapted. Light availability appears to be the critical variable for the development of hotspot phytoplankton blooms, which in turn supports the highly productive regional food web.

The West Antarctic Peninsula (WAP) is a highly productive polar ecosystem where phytoplankton dynamics are regulated by intense bottom‐up control from light and iron availability. Rapid climate change along the WAP is driving shifts in the mixed layer depth and iron availability. Elucidating the relative role of each of these controls and their interactions is crucial for understanding of how primary productivity will change in coming decades. Using a combination of ultra‐high‐resolution variable chlorophyll fluorescence together with fluorescence lifetime analyses on the 2017 Palmer Long Term Ecological Research cruise, we mapped the temporal and spatial variability in phytoplankton photophysiology across the WAP. Highest photosynthetic energy conversion efficiencies and lowest fluorescence quantum yields were observed in iron replete coastal regions. Photosynthetic energy conversion efficiencies decreased by ~ 60% with a proportional increase in quantum yields of thermal dissipation and fluorescence on the outer continental shelf and slope. The combined analysis of variable fluorescence and lifetimes revealed that, in addition to the decrease in the fraction of inactive reaction centers, up to 20% of light harvesting chlorophyll‐protein antenna complexes were energetically uncoupled from photosystem II reaction centers in iron‐limited phytoplankton. These biophysical signatures strongly suggest severe iron limitation of photosynthesis in the surface waters along the continental slope of the WAP.

Amundsen Sea polynyas are among the most productive, yet climate-sensitive ecosystems in the Southern Ocean and host massive annual phytoplankton blooms. These blooms are believed to be controlled by iron fluxes from melting ice and icebergs and by intrusion of nutrient-rich Circumpolar Deep Water, however the interplay between iron effects and other controls, such as light availability, has not yet been quantified. Here, we examine phytoplankton photophysiology in relation to Fe stress and physical forcing in two largest polynyas, Amundsen Sea Polynya (ASP) and Pine Island Polynya (PIP), using the combination of high-resolution variable fluorescence measurements, fluorescence lifetime analysis, photosynthetic rates, and Fe-enrichment incubations. These analyses revealed strong Fe stress in the ASP, whereas the PIP showed virtually no signatures of Fe limitation. In spite of enhanced iron availability in the PIP, chlorophyll biomass remained ∼ 30–50% lower than in the Fe-stressed ASP. This apparent paradox would not have been observed if iron were the main control of phytoplankton bloom in the Amundsen Sea. Long-term satellite-based climatology records revealed that the ASP is exposed to significantly higher solar irradiance levels throughout the summer season, as compared to the PIP region, suggesting that light availability controls the magnitude of phytoplankton blooms in the Amundsen Sea. Our data suggests that higher Fe availability (e.g., due to higher melting rates of ice sheets) would not necessarily increase primary productivity in this region. Furthermore, stronger wind-driven vertical mixing in expanding ice-free areas may lead to reduction in light availability and productivity in the future.

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10-5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

To assess the role of redox state of photosystem II (PSII) acceptor side electron carriers in PSII photochemical activity, we studied sub-millisecond fluorescence kinetics of the wild type Synechocystis PCC 6803 and its mutants with natural variability in the redox state of the plastoquinone (PQ) pool. In cyanobacteria, dark adaptation tends to reduce PQ pool and induce a shift of the cyanobacterial photosynthetic apparatus to State 2, whereas illumination oxidizes PQ pool, leading to State 1 (Mullineaux, C. W., and Holzwarth, A. R. (1990) FEBS Lett., 260, 245-248). We show here that dark-adapted Ox(-) mutant with naturally reduced PQ is characterized by slower QA(-) reoxidation and O2 evolution rates, as well as lower quantum yield of PSII primary photochemical reactions (Fv/Fm) as compared to the wild type and SDH(-) mutant, in which the PQ pool remains oxidized in the dark. These results indicate a large portion of photochemically inactive PSII reaction centers in the Ox(-) mutant after dark adaptation. While light adaptation increases Fv/Fm in all tested strains, indicating PSII activation, by far the greatest increase in Fv/Fm and O2 evolution rates is observed in the Ox(-) mutant. Continuous illumination of Ox(-) mutant cells with low-intensity blue light, that accelerates QA(-) reoxidation, also increases Fv/Fm and PSII functional absorption cross-section (590 nm); this effect is almost absent in the wild type and SDH(-) mutant. We believe that these changes are caused by the reorganization of the photosynthetic apparatus during transition from State 2 to State 1. We propose that two processes affect the PSII activity during changes of light conditions: 1) reversible inactivation of PSII, which is associated with the reduction of electron carriers on the PSII acceptor side in the dark, and 2) PSII activation under low light related to the increase in functional absorption cross-section at 590 nm.

Using solar energy suboptimally How efficient are phytoplankton at converting sunlight into the products of photosynthesis? The two other pathways that that absorbed energy can take are emission back to the environment by fluorescence or conversion to heat. Lin et al. measured phytoplankton fluorescence lifetimes in the laboratory and combined them with satellite measurements of variable chlorophyll fluorescence. Combined, they determined the quantum yields of photochemistry and fluorescence in four ocean basins. Approximately 60% of absorbed solar energy is converted to heat, a figure 50% higher than has been determined for conditions of optimal growth. Science , this issue p. 264

The algorithm for estimation of the quantum yield of phytoplankton fluorescence from the remote sensing satellite of the MODIS tool is discussed in the paper. There is an example of manifestation of the iron limitation in the Amundsen Sea. Amundsen Sea encloses two polynyas: Fe-limited ASP (Amundsen Sea Polynya) and Fe-replete PIP (Pine Island Polynya). We present a procedure for comparing the mean values of the quantum yield of phytoplankton fluorescence in these regions and the requirements for it. To meet these requirements, the data of two satellite systems were analyzed and compared: MODIS and AVHRR. Analysis of the data made it possible to observe the differences in the mean values of the quantum yield of phytoplankton fluorescence in these two regions during the bloom period of the Amundsen Sea phytoplankton in 2012.

To better understand how photosystem (PS) activity is regulated during state transitions in cyanobacteria, we studied photosynthetic parameters of photosystem II (PSII) and photosystem I (PSI) in Synechocystis PCC 6803 wild type (WT) and its mutants deficient in oxidases (Ox(-)) or succinate dehydrogenase (SDH(-)). Dark-adapted Ox(-) mutant, lacking the oxidation agents, is expected to have a reduced PQ pool, while in SDH(-) mutant the PQ pool after dark adaptation will be more oxidized due to partial inhibition of the respiratory chain electron carriers. In this work, we tested the hypothesis that control of balance between linear and cyclic electron transport by the redox state of the PQ pool will affect PSII photosynthetic activity during state transition. We found that the PQ pool was reduced in Ox(-) mutant, but oxidized in SDH(-) mutant after prolonged dark adaptation, indicating different states of the photosynthetic apparatus in these mutants. Analysis of variable fluorescence and 77K fluorescence spectra revealed that the WT and SDH(-) mutant were in State 1 after dark adaptation, while the Ox(-) mutant was in State 2. State 2 was characterized by ~1.5 time lower photochemical activity of PSII, as well as high rate of P700 reduction and the low level of P700 oxidation, indicating high activity of cyclic electron transfer around PSI. Illumination with continuous light 1 (440 nm) along with flashes of light 2 (620 nm) allowed oxidation of the PQ pool in the Ox(-) mutant, thus promoting it to State 1, but it did not affect PSII activity in dark adapted WT and SDH(-) mutant. State 1 in the Ox(-) mutant was characterized by high variable fluorescence and P700(+) levels typical for WT and the SDH(-) mutant, indicating acceleration of linear electron transport. Thus, we show that PSII of cyanobacteria has a higher photosynthetic activity in State 1, while it is partially inactivated in State 2. This process is controlled by the redox state of PQ in cyanobacteria through enhancement/inhibition of electron transport on the acceptor side of PSII.

Diatoms, unicellular phytoplankton that account for ∼40% of marine primary productivity, often dominate coastal and open-ocean upwelling zones. Limitation of growth and productivity by iron at low light is attributed to an elevated cellular Fe requirement for the synthesis of Fe-rich photosynthetic proteins. In the dynamic coastal environment, Fe concentrations and daily surface irradiance levels can vary by two to three orders of magnitude on short spatial and temporal scales. Although genome-wide studies are beginning to provide insight into the molecular mechanisms used by diatoms to rapidly respond to such fluxes, their functional role in mediating the Fe stress response remains uncharacterized. Here, we show, using reverse genetics, that a death-specific protein (DSP; previously named for its apparent association with cell death) in the coastal diatom Thalassiosira pseudonana (TpDSP1) localizes to the plastid and enhances growth during acute Fe limitation at subsaturating light by increasing the photosynthetic efficiency of carbon fixation. Clone lines overexpressing TpDSP1 had a lower quantum requirement for growth, increased levels of photosynthetic and carbon fixation proteins, and increased cyclic electron flow around photosystem I. Cyclic electron flow is an ATP-producing pathway essential in higher plants and chlorophytes with a heretofore unappreciated role in diatoms. However, cells under replete conditions were characterized as having markedly reduced growth and photosynthetic rates at saturating light, thereby constraining the benefits afforded by overexpression. Widespread distribution of DSP-like sequences in environmental metagenomic and metatranscriptomic datasets highlights the presence and relevance of this protein in natural phytoplankton populations in diverse oceanic regimes.

Metal toxicity affects a myriad of physiological and metabolic processes, including photosynthesis, in plant cells; however, the primary sites of metal poisoning and the sequence of physiological alterations remain a topic of controversy. Using a fluorescence induction and relaxation technique and quantitative immunoblots, we examined toxic effects of metal ions (Cu, Zn, Cd and Pb) on photosynthetic light-harvesting processes, photochemistry in photosystem II (PSII), and photosynthetic electron transport in symbiotic dinoflagellates, Symbiodinium spp. (zooxanthellae). The analysis of metal-induced alterations in fluorescence parameters revealed an early inhibition of the electron transport between PSII and PSI and of the maximum rates of photosynthetic electron transport ( P max ), suggesting that the primary targets of metal toxicity are the processes downstream PSII, rather than photochemistry in PSII. The Cu-, Zn-, and Cd-induced inhibition of electron transport between PSII and PSI was followed by a decrease in the energy transfer in light-harvesting complexes, implying that these metals may impact the functional integrity of lipid membranes. A striking decrease in P max was observed much earlier than any alterations in photochemistry or time constants for electron transport within PSII and occurred prior to a decrease in cellular Rubisco content. This is common for both essential (Cu and Zn) and non-essential (Cd and Pb) metals. However, Cu and Zn have a greater impact on photosynthetic processes, while Cd and Pb affect cell growth rates to a greater extent. Immunoblot protein analysis revealed that PSII core proteins, PsbA and PsbD, start to degrade prior to Rubisco and ATP synthase under exposure to Zn, Cd, or Pb. In contrast, Cu poisoning leads to stronger degradation of Rubisco and ATP synthase than of PsbA and PsbD. High growth irradiance accelerated the damage to the electron transport between PSII and PSI and photochemistry in PSII. Our results are important for understanding the physiological processes involved in metal poisoning in aquatic organisms and provide a background for the development of express diagnostics and identification of stressors in aquatic environments.

[1] We describe variability in the phytoplankton physiological status in the west Scotia Sea of the Southern Ocean in the early austral summer prior to and at the beginning of a phytoplankton bloom. This area is characterized by high concentrations of major nutrients (such as nitrate, phosphate, and silicate), but exhibit chronically low concentrations of dissolved iron and deep vertical mixing. Using a fast repetition rate fluorometry, we measured photosynthetic characteristics of phytoplankton in the euphotic zone. These measurements provide an express diagnostic of the effects of environmental factors, including iron limitation, on photosynthetic processes. The quantum yields of photochemistry in Photosystem II (Fv/Fm) in subsurface phytoplankton were, on average, 40% lower than the maximum values for nutrient-replete communities. Higher values of Fv/Fm were observed in the frontal mixing zone that may have been caused by the induction of iron into the euphotic layer. Our results suggest physiological signatures of iron limitation of photosynthesis in the western Scotia Sea in the early austral summer. The data imply that, together with light conditions and grazing pressure, iron availability may be important for phytoplankton growth in the Scotia Sea even in the early summer.

The integration of a FIRe sensor into a glider allows us to map, with high temporal and spatial resolution, phytoplankton physiological responses to physical forcing. Different missions were designed to evaluate the temporal and spatial variability of phytoplankton physiology by using a drift and a station keeping mission, respectively. Diel cycles collected show a clear diurnal variations driven by incident radiation, with both maximal fluorescence and photosynthetic efficiency (in any light adapted phytoplankton) showing reduced values only in the upper 10–15 meters of the water column at the highest irradiances. Further analyses comparing different MLD regimes have shown different photoacclimation responses (light saturation parameter, Ek) resulting from differences in solar radiation exposure conditions (both time and intensity), reflected in the depth of the ML. Further analyses include determining a method to correct the FIRe glider fluorescence profiles in the upper ocean during daytime by comparing the maximum fluorescence during the highest irradiance (daytime) with the lowest irradiance (nighttime).

Using a novel, pulsed micro-second time-resolved photoacoustic (PA) instrument, we measured thermal dissipation and energy storage (ES) in the intact cells of wild type (WT) Chlamydomonas reinhardtii, and mutants lacking either PSI or PSII reaction centers (RCs). On this time scale, the kinetic contributions of the thermal expansion component due to heat dissipation of absorbed energy and the negative volume change due to electrostriction induced by charge separation in each of the photosystems could be readily distinguished. Kinetic analysis revealed that PSI and PSII RCs exhibit strikingly different PA signals where PSI is characterized by a strong electrostriction signal and a weak thermal expansion component while PSII has a small electrostriction component and large thermal expansion. The calculated ES efficiencies at ~10 μs were estimated to be 80 ± 5 and 50 ± 13% for PSII-deficient mutants and PSI-deficient mutants, respectively, and 67 ± 2% for WT. The overall ES efficiency was positively correlated with the ratio of PSI to PSI + PSII. Our results suggest that the shallow excitonic trap in PSII limits the efficiency of ES as a result of an evolutionary frozen metabolic framework of two photosystems in all oxygenic photoautotrophs.

During the past several decades, numerous reports from disparate geographical areas have documented an increased frequency of “bleaching” in reef-forming corals. The phenomenon, triggered by increased sea surface temperatures, occurs when the cnidarian hosts digest and/or expel their intracellular, photosynthetic dinoflagellate symbionts (“zooxanthellae” in the genus Symbiodinium ). Although coral bleaching is often followed by the death of the animal hosts, in some cases, the animal survives and can be repopulated with viable zooxanthellae. The physiological factors determining the ability of the coral to survive bleaching events are poorly understood. In this study, we experimentally established that bleaching and death of the host animal involve a caspase-mediated apoptotic cascade induced by reactive oxygen species produced primarily by the algal symbionts. In addition, we demonstrate that, although some corals naturally suppress caspase activity and significantly reduce caspase concentration under high temperatures as a mechanism to prevent colony death from apoptosis, even sensitive corals can be prevented from dying by application of exogenous inhibitors of caspases. Our results indicate that variability in response to thermal stress in corals is determined by a four-element, combinatorial genetic matrix intrinsic to the specific symbiotic association. Based on our experimental data, we present a working model in which the phenotypic expression of this symbiont/host relationship places a selective pressure on the symbiotic association. The model predicts the survival of the host animals in which the caspase-mediated apoptotic cascade is down-regulated.

By using a nondestructive, ultrasensitive, fluorescence kinetic technique, we measure in situ the photochemical energy conversion efficiency and electron transfer kinetics on the acceptor side of histidine-tagged photosystem II core complexes tethered to gold surfaces. Atomic force microscopy images coupled with Rutherford backscattering spectroscopy measurements further allow us to assess the quality, number of layers, and surface density of the reaction center films. Based on these measurements, we calculate that the theoretical photoelectronic current density available for an ideal monolayer of core complexes is 43 microA cm(-2) at a photon flux density of 2000 micromol quanta m(-2) s(-1) between 365 and 750 nm. While this current density is approximately two orders of magnitude lower than the best organic photovoltaic cells (for an equivalent area), it provides an indication for future improvement strategies. The efficiency could be improved by increasing the optical cross section, by tuning the electron transfer physics between the core complexes and the metal surface, and by developing a multilayer structure, thereby making biomimetic photoelectron devices for hydrogen generation and chemical sensing more viable.

In this paper we summarise the photo-physiological responses of phytoplankton to upwelling of macronutrients in mesoscale eddies in the subtropical North Atlantic (EDDIES project, Sargasso Sea) and subtropical North Pacific (E-FLUX project, Hawaii). The observations, obtained on two sets of cruises over 2 years, occupied six cyclonic eddies and two mode-water eddies. The photosynthetic physiological parameters were measured using a bench-top fluorescence induction and relaxation (FIRe) system and a submersible in situ fast repetition rate fluorometer (FRRF) deployed on an undulating towed vehicle. Both of these instruments were used to provide highly sensitive and well-resolved data on community responses. The responses are dependent on both the type of eddy and its stage of development. Our results indicate that, while cyclonic eddies in the Atlantic and Pacific can increase primary photosynthetic production early in their development, mode-water eddies in the subtropical North Atlantic can support patchy blooms of large diatoms for long periods of time (more than 3 months).

When diatoms are stressed for inorganic nitrogen they remodel their intermediate metabolism and redirect carbon towards lipid biosynthesis. However, this response comes at a significant cost reflected in decreased photosynthetic energy conversion efficiency and growth. Here we explore a molecular genetics approach to restrict the assimilation of inorganic nitrogen by knocking down nitrate reductase (NR). The transformant strain, NR21, exhibited about 50% lower expression and activity of the enzyme but simultaneously accumulated over 40% more fatty acids. However, in contrast to nitrogen-stressed wild-type (WT) cells, which grow at about 20% of the rate of nitrogen-replete cells, growth of NR21 was only reduced by about 30%. Biophysical analyses revealed that the photosynthetic energy conversion efficiency of photosystem II was unaffected in NR21; nevertheless, the plastoquinone pool was reduced by 50% at the optimal growth irradiance while in the WT it was over 90% oxidized. Further analyses reveal a 12-fold increase in the glutamate/glutamine ratio and an increase NADPH and malonyl-CoA pool size. Transcriptomic analyses indicate that the knock down resulted in changes in the expression of genes for lipid biosynthesis, as well as the expression of specific transcription factors. Based on these observations, we hypothesize that the allocation of carbon and reductants in diatoms is controlled by a feedback mechanism between intermediate metabolites, the redox state of the plastid and the expression and binding of transcription factors related to stress responses.

To prevent photooxidative damage under supraoptimal light, photosynthetic organisms evolved mechanisms to thermally dissipate excess absorbed energy, known as non-photochemical quenching (NPQ). Here we quantify NPQ-induced alterations in light-harvesting processes and photochemical reactions in Photosystem 2 (PS2) in the pennate diatom Phaeodactylum tricornutum. Using a combination of picosecond lifetime analysis and variable fluorescence technique, we examined the dynamics of NPQ activation upon transition from dark to high light. Our analysis revealed that NPQ activation starts with a 2-3-fold increase in the rate constant of non-radiative charge recombination in the reaction center (RC); however, this increase is compensated with a proportional increase in the rate constant of back reactions. The resulting alterations in photochemical processes in PS2 RC do not contribute directly to quenching of antenna excitons by the RC, but favor non-radiative dissipation pathways within the RC, reducing the yields of spin conversion of the RC chlorophyll to the triplet state. The NPQ-induced changes in the RC are followed by a gradual ~ 2.5-fold increase in the yields of thermal dissipation in light-harvesting complexes. Our data suggest that thermal dissipation in light-harvesting complexes is the major sink for NPQ; RCs are not directly involved in the NPQ process, but could contribute to photoprotection via reduction in the probability of (3)Chl formation.

Solar radiation absorbed by marine phytoplankton can follow three possible paths. By simultaneously measuring the quantum yields of photochemistry and chlorophyll fluorescence in situ, we calculate that, on average, ~60% of absorbed photons are converted to heat, only 35% are directed toward photochemical water splitting, and the rest are reemitted as fluorescence. The spatial pattern of fluorescence yields and lifetimes strongly suggests that photochemical energy conversion is physiologically limited by nutrients. Comparison of in situ fluorescence lifetimes with satellite retrievals of solar-induced fluorescence yields suggests that the mean values of the latter are generally representative of the photophysiological state of phytoplankton; however, the signal-to-noise ratio is unacceptably low in extremely oligotrophic regions, which constitute 30% of the open ocean.

The availability of iron is known to exert a controlling influence on biological productivity in surface waters over large areas of the ocean and may have been an important factor in the variation of the concentration of atmospheric carbon dioxide over glacial cycles. The effect of iron in the Southern Ocean is particularly important because of its large area and abundant nitrate, yet iron-enhanced growth of phytoplankton may be differentially expressed between waters with high silicic acid in the south and low silicic acid in the north, where diatom growth may be limited by both silicic acid and iron. Two mesoscale experiments, designed to investigate the effects of iron enrichment in regions with high and low concentrations of silicic acid, were performed in the Southern Ocean. These experiments demonstrate iron's pivotal role in controlling carbon uptake and regulating atmospheric partial pressure of carbon dioxide.

In oxygenic photosynthesizing organisms, it has been noticed on a number of occasions that photosynthetic performance was lower in the afternoon than in the morning, at the same light intensities. This hysteresis phenomenon is called the 'afternoon depression' and has been observed in phytoplankton, macroalgae, and higher plants. Here we characterize, with high temporal resolu- tion, in situ diel courses of oxygen evolution and chlorophyll fluorescence yields in 3 Indo-Pacific corals (Favia favus, Goniopora lobata, Plerogyra sinuosa) and 2 Caribbean symbiotic corals (Montas- trae faveolata and Porites astreoides) using a 3-chamber submersible respirometer and a SCUBA- based fast repetition rate fluorometer. In contrast to all previously published cases, the oxygen mea- surements revealed an unexpected hysteresis, with higher photosynthetic rates occurring in the afternoon than in the morning. The oxygen diel patterns were highly consistent in all organisms examined, including the 3 corals and 2 macroalgae (Ulva sp. and Gracilaria sp.) from the Red Sea. Surprisingly, the diurnal patterns of the quantum yields of photochemistry in Photosystem II (PSII), assessed from variable fluorescence (quantum yield of photochemistry in PSII measured under ambi- ent light, ∆F '/Fm') exhibited much higher variability and often showed a hysteresis pattern opposite to that of oxygen. In most organisms the values of ∆F '/Fm' and the deduced rates of photosynthetic elec- tron transport were higher in the morning than in the afternoon; however, the opposite trend with lower values of ∆F '/Fm' in the morning was also observed. Lower values of ∆F '/Fm' were always accompanied by higher quantum yields of non-photochemical quenching, consistent with the ener- getic balance within the primary photosynthetic reactions. The direction of the diurnal hysteresis in variable fluorescence appears to be species-specific and may vary even within the same species, reflecting microscale variability in bio-optical properties and gross photosynthesis of the corals.

Fluorescent pigments in several Indo-Pacific and Caribbean anthozoans have recently been identified as proteins related to the Green-fluorescent protein (GFP) of the hydromedusa Aequorea victoria. Here we show that GFP is widely distributed in many Caribbean species. The fluorescence excitation and emission spectra for the pigment are similar to those reported elsewhere for coral and noncoral GFP and the fluorescence quantum yield is estimated to be 35%. Spectral and molecular characterization of the isolated protein clearly show it to be GFP, and laboratory and in situ fluorescence measurements and Western blot analysis show that it is widespread. Bathymetric studies of GFP content using Western blots for the ecologically important congeneric corals Montastraea faveolata and Montastraea cavernosa show that there is no significant correlation between depth and GFP concentration. Nucleotide sequence data of GFP from M. faveolata and M. cavernosa show 88.2% sequence homology with each other and 46.4% homology with A. victoria GFP, whereas the percent homology with A. victoria at the amino acid level was 31.1 and 28.4% for M. cavernosa and M. faveolata, respectively, and 82.7% with each other. Measurements of reflectance and of the excitation spectrum for chlorophyll fluorescence in GFP-containing corals indicate that GFP absorption, emission, and reflection have negligible impact on the level of solar radiation reaching the zooxanthellae and therefore play no role in coral photosynthesis by either addition or removal of photons.

EisenEx�the second in situ iron enrichment experiment in the Southern Ocean�was performed in the Atlantic sector over 3 weeks in November 2000 with the overarching goal to test the hypothesis that primary productivity in the Southern Ocean is limited by iron availability in the austral spring. Underwater irradiance, chlorophyll a (Chl a), photochemical efficiency, and primary productivity were measured inside and outside of an iron-enriched patch in order to quantify the response of phytoplankton to iron fertilization. Chl a concentration and photosynthetic rate (14C uptake in simulated in situ incubations) were measured in pico-, nano-, and microphytoplankton. Photochemical efficiency was studied with fast repetition rate fluorometry and xenon-pulse amplitude modulated fluorometry. The high-nutrient low-chlorophyll waters outside the Fe-enriched patch were characterized by deep euphotic zones (63-72 m), low Chl a (48-56 mg m-2), low photosynthetic efficiency (Fv/Fm ~ 0.3), and low daily primary productivity (130-220 mg C m-2 d-1). Between 70 and 90% of Chl a was found in pico- and nanophytoplankton. During the induced bloom, Fv/Fm increased up to ;0.55, primary productivity and Chl a reached the maximum values of 790 mg C m-2 d-1 and 231 mg Chl a m-2, respectively. As a consequence, the euphotic depth decreased to ~41 m. Picophytoplankton biomass hardly changed. Nano- and microphytoplankton biomass increased. In the first 2 weeks of the experiment, when the depth of the upper mixed layer was mostly 80 m.

In many species of symbiotic corals, spawning occurs synchronously several nights after the full moon. This process is correlated with the level of lunar irradiance, but the mechanism by which these cnidarian/zooxanthellate symbioses can detect such low levels of light remains unknown. Here we report the first biophysical evidence that the host animal exhibits extraordinarily sensitive photoreception in the blue region of the spectrum. Using a high-resolution laser-induced signal to detect tentacle scattering, we measured the effect of low irradiance on the contraction of polyps in the corals that normally have their tentacles extended in darkness. Similar to most deep-sea invertebrates, the action spectra of coral photoreception reveal a maximum sensitivity in the blue, at 480 nm, with a spectral band width (at full-width half-maximum) of ca. 110 nm. The spectra closely overlap the maximal transparency of oligotrophic tropical waters, thus optimizing the perception of low light at depth. The detected threshold of photoreception sensitivity is ∼1.2 X 10 15 quanta m -2 s -1 in the blue region. This makes corals capable of sensing the blue portion of lunar irradiance, as evidenced from the recorded slight contractions of polyp tentacles under variations in moonlight intensity.

In zooxanthellate corals, excess excitation energy can be dissipated as heat (nonphotochemical quenching), thereby providing protection against oxidative damage by supraoptimal light in shallow reefs. To identify and quantify the photoprotective mechanisms, we studied the diel variability of chlorophyll fluorescence yields and photosynthetic parameters in situ in corals, using moored and SCUBA-based fast-repetition-rate fluorometers. The results reveal that nonphotochemical quenching is triggered prior to saturation of photosynthetic electron transport by downregulation of the reaction centers of Photosystem II (PSII). This process dissipates up to 80% of the excitation energy. On a sunny day in shallow waters, the daily integrated flux of photons absorbed, and subsequently dissipated as heat, is ;4 times that used for photosynthesis. Fluorescence quenching is further accompanied by a slight reduction in the functional absorption cross section for PSII that results from thermal dissipation of excitation energy in the light-harvesting antennae. These two processes are highly dynamic and adjust to irradiance changes on timescales consistent with the passage of clouds across the sky. Under supraoptimal irradiance, however, up to 30% of PSII reaction centers become photoinhibited, and these are repaired only after several hours of low irradiance. In shallow corals, between 10% and 20% of the reactions centers are chronically photoinhibited and appear to remain permanently nonfunctional throughout the year. Our results establish, for the first time, the suite of biophysical mechanisms that optimize photosynthesis while simultaneously providing photoprotection in symbiotic corals in situ.

A newly developed underwater fast repetition rate fluorometer (FRRF) was used for in situ measurements of chlorophyll fluorescence yields on the reef-building corals Montastraea faveo- lata and Montastraea cavernosa from around Lee Stocking Island, Bahamas. Diel studies of the quan- tum yield of chlorophyll fluorescence (⌉F '/ Fm') in photosystem II (PSII) reveal a pattern of mid-day depression of ⌉F '/ Fm' in both of these species of coral. At the same time, non-photochemical quench- ing (qN ) increased significantly during the day, a pattern consistent with the regulation of PSII by dynamic photoinhibition mediated by non-photochemical quenching. Despite these mid-day depres- sions in ⌉F '/ Fm', net productivity, measured as oxygen flux, remains high, suggesting that non-pho- tochemical quenching dissipates the majority of the absorbed photons at mid-day and protects the photosynthetic apparatus, allowing the endosymbiotic dinoflagellates (zooxanthellae) to operate at maximum rates of photosynthesis. In 1999 measurements of ⌉F '/ Fm' on M. faveolata over a bathy- metric range of 2 to 30 m showed an increase in ⌉F '/ Fm' with increasing depth when measured at the same time of day. This suggests, although there is year-to-year variability, that changes in the under- water light field, and photoacclimation to that light field, control the degree of photoprotection attrib- utable to non-photochemical quenching in the zooxanthellae of these corals. The fluorescence yields of M. faveolata exposed to elevated temperatures (> 32°C) in the field showed a significant decrease in ⌉F '/Fm' before visible signs (e.g., paling of colonies) occurred. It was also possible to predict which colonies at the same depth and light regime would bleach first in response to elevated temperatures before any visible signs of bleaching were evident using ⌉F '/Fm' as a predictor.

Recently, an underwater version of a fast repetition rate fluorometer (FRRF) was developed for the non-destructive study of fluorescence yields in benthic photoautotrophs. We used an FRRF to study bleached colonies of the corals, Montastraea faveolata and Diploria labyrinthiformes at sites surrounding Lee Stocking Island, Exuma, Bahamas, to assess their recovery from bleaching (∼1 year after the initial bleaching event) induced by elevated temperatures. The steady state quantum yields of chlorophyll a fluorescence (Δ F ′/ F ′ m ) from photosystem II (PSII) within coral colonies were separated into three categories representing visibly distinct degrees of bleaching ranging from no bleaching to completely bleached areas. Differences in Δ F ′/ F ′ m were significantly different from bleached to unbleached regions within colonies. Dark, unbleached regions within colonies exhibited significantly higher Δ F ′/ F ′ m values (0.438±0.019; mean±S.D.) when compared to lighter regions, and occupied a majority of the colonies’ surface area (46–73%). Bleached regions exhibited significantly lower Δ F ′/ F ′ m (0.337±0.014) and covered only 7–25% of the colonies’ surface area. The observations from this study suggest that zooxanthellae in bleached regions of a colony exhibit reduced photosynthetic activity as long as one year after a bleaching event and that in situ fluorescence techniques such as FRRF are an effective means of studying coral responses and recovery from natural or anthropogenic stress in a non-destructive manner.

Benthic photoautotrophic organisms significantly contribute to the productivity of shallow tropical coastal ecosystems. However, measurements of photosynthetic light use and dissipation in benthic organisms are complicated by taxonomic diversity, spatial heterogeneity, natural variability in local nutrient, irradiance, and temperature regimes, as well as destructive sampling protocols. To help overcome these problems, we developed a SCUBA-based fast repetition rate (FRR) fluorometer for measurements of variable chlorophyll fluorescence in corals, sea grasses, macroalgae, and algal turfs. Photosynthetic light use and electron transport can be readily calculated from variable fluorescence kinetics. Using the SCUBA-based FRR fluorometer, changes in photosynthetic processes can be measured nondestructively in situ with high spatial and temporal resolution. Here we describe the instrument design and characteristics and present representative field results.

A Single Cell Fast Repetition Rate (SCFRR) fluorometer was developed to measure the quantum yield of photochemistry, the functional absorption cross section of PS II and the kinetics of electron transport on the acceptor side of PS II in individual algal cells. These parameters are used to quantify the cell-specific photosynthetic performance in natural phytoplankton assembledges in aquatic ecosystems. The SCFRR technique measures chlorophyll fluorescence transients induced by a precisely controlled series of excitation flashlets that cumulatively saturate PS II within 120 μs. To meet the requirement in the analysis for single algal cells, the measurements are conducted in micro volumes, such that the probability of probing more than one cell at a time is vanishingly low. We designed a novel, computer-controlled hydromechanical system to deliver a portion of the sample into the measuring chamber and, following measurement, remove it into one of six sorting containers. The fluorescence signal is induced by a series of high frequency flashlets obtained from high luminosity blue light-emitting diodes and is acquired by a novel red-sensitive PMT-based detection system exhibiting both high sensitivity and a very wide dynamic range. The wide dynamic range of the detector allows SCFRR measurements for a wide variety of cell sizes ranging from 1 to 100 μm equivalent spherical diameter. The compact and light-weight design makes the SCFRR Fluorometer applicable for both laboratory and field studies.

We use an advanced fluorescence method of Nonlinear Laser Fluorimetry in combination with Fluorescence Induction and Relaxation technique to study the influence of excess-light conditions on the physiological state of the green alga Chlorella pyrenoidosa. We demonstrate that zeaxanthin-dependent non-photochemical quenching leads to a significant increase in the rate constant of singlet-singlet annihilation of chlorophyll a excited state, which suggests profound conformational changes in the light-harvesting complexes of photosystem II.

The development of advanced technologies for environmental monitoring of benthic communities under DoD jurisdiction requires an understanding of how different environmental factors affect the key elements of the ecosystems and the selection of specific monitoring protocols that are most appropriate for the identification of particular stressors.

In this studies we used classical and laser methods for cyanobacteria diagnostics: Fluorescence Induction and Relaxation and Non-Linear Laser Fluorimetry in order to obtain the whole set of photophysical parameters of cyanobacteria. Different photophysical processes that take place in photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC6803 were studied with mentioned above fluorescent methods and sets of photophysical parameters were determined. The results allow us to suggest a model of photo adaptation processes under excess irradiance (depending on light intensity and spectrum).

The newest approach in the saturation fluorimetry of photosynthetic organisms by the example of phytoplankton was developed. The theoretical model and the inverse problem of the saturation fluorimetry are discussed. The results of evaluation of molecular photophysical parameters of alga Chlorella pyrenoidosa under various stress factors, such as presence of DCMU and Cu2+ ions are presented. The correlation between theese parameters and the parameters obtained using Fluorescence Induction and Relaxation technique is discussed.

The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: ( i ) an organic ECM, which facilitates cell–cell and cell–substrate adhesion; ( ii ) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and ( iii ) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft ( Xenia elongata ) and hard ( Montipora digitata ) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata , and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata . Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of 45 Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization.

Over the past three decades, massive bleaching events of zooxanthellate corals have been documented across the range of global distribution. Although the phenomenon is correlated with relatively small increases in sea-surface temperature and enhanced light intensity, the underlying physiological mechanism remains unknown. In this article we demonstrate that thylakoid membrane lipid composition is a key determinate of thermal-stress sensitivity in symbiotic algae of cnidarians. Analyses of thylakoid membranes reveal that the critical threshold temperature separating thermally tolerant from sensitive species of zooxanthellae is determined by the saturation of the lipids. The lipid composition is potentially diagnostic of the differential nature of thermally induced bleaching found in scleractinian corals. Measurements of variable chlorophyll fluorescence kinetic transients indicate that thermally damaged membranes are energetically uncoupled but remain capable of splitting water. Consequently, a fraction of the photosynthetically produced oxygen is reduced by photosystem I through the Mehler reaction to form reactive oxygen species, which rapidly accumulate at high irradiance levels and trigger death and expulsion of the endosymbiotic algae. Differential sensitivity to thermal stress among the various species of Symbiodinium seems to be distributed across all clades. A clocked molecular phylogenetic analysis suggests that the evolutionary history of symbiotic algae in cnidarians selected for a reduced tolerance to elevated temperatures in the latter portion of the Cenozoic.

Colonies of the Caribbean coral Montastraea cavernosa exhibit a solar-stimulated orange-red fluorescence that is spectrally similar to a variety of fluorescent proteins expressed by corals. The source of this fluorescence is phycoerythrin in unicellular, nonheterocystis, symbiotic cyanobacteria within the host cells of the coral. The cyanobacteria coexist with the symbiotic dinoflagellates (zooxanthellae) of the coral and express the nitrogen-fixing enzyme nitrogenase. The presence of this prokaryotic symbiont in a nitrogen-limited zooxanthellate coral suggests that nitrogen fixation may be an important source of this limiting element for the symbiotic association.

LONG-TERM GOALS. The long term goals of this research program are to develop the biophysical tools and models that predict the sources of variability of chlorophyll fluorescence from benthic targets in shallow waters. OBJECTIVES. The scientific objectives of the project are to relate the fluorescence lifetimes of photosynthetic targets to the amplitude-based fluorescence measurements. In so doing, laser-based fluorescence retrievals from line-scanners or other in situ instruments can be deconvoluted in the lifetime domain to reconstruct biophysical and physiological information about the photosynthetic activity of target organisms. APPROACH. The basic approach taken is to compare high-precision, laser-induced fluorescence lifetimes of model organisms with amplitude based measurements using fast-repetition rate fluorescence techniques. The research is conducted in collaboration with Drs. Maxim Gorbunov, Zbigniew Kolber and Edward Castner. Dr. Castner is a physical chemist specializing in photochemical processes in the Department of Chemistry at Brookhaven National Laboratory. The model organisms used are primarily cultured zooxanthellae obtained from a variety of symbiotic marine invertebrates.