Using picosecond fluorescence lifetime analysis to determine photosynthesis in the world's oceans.

Phytoplankton in the ocean account for less than 1% of the global photosynthetic biomass, but contribute about 45% of the photosynthetically fixed carbon on Earth. This amazing production/biomass ratio implies a very high photosynthetic efficiency. But, how efficiently is the absorbed light used in marine photosynthesis? The introduction of picosecond and then femtosecond lasers for kinetic measurements in mid 1970s to 90 s was a revolution in basic photosynthesis research that vastly improved our understanding of the energy conversion processes in photosynthetic reactions. Until recently, the use of this technology in the ocean was not feasible due to the complexity of related instrumentation and the lack of picosecond lasers suitable for routine operation in the field. However, recent advances in solid-state laser technology and the development of compact data acquisition electronics led to the application of picosecond fluorescence lifetime analyses in the field. Here, we review the development of operational ultrasensitive picosecond fluorescence instruments to infer photosynthetic energy conversion processes in ocean ecosystems. This analysis revealed that, in spite of the high production/biomass ratio in marine phytoplankton, the photosynthetic energy conversion efficiency is exceptionally low-on average, ca. 50% of its maximum potential, suggesting that most of the contemporary open ocean surface waters are extremely nutrient deficient.

Salp blooms drive strong increases in passive carbon export in the Southern Ocean.

The Southern Ocean contributes substantially to the global biological carbon pump (BCP). Salps in the Southern Ocean, in particular Salpa thompsoni, are important grazers that produce large, fast-sinking fecal pellets. Here, we quantify the salp bloom impacts on microbial dynamics and the BCP, by contrasting locations differing in salp bloom presence/absence. Salp blooms coincide with phytoplankton dominated by diatoms or prymnesiophytes, depending on water mass characteristics. Their grazing is comparable to microzooplankton during their early bloom, resulting in a decrease of ~1/3 of primary production, and negative phytoplankton rates of change are associated with all salp locations. Particle export in salp waters is always higher, ranging 2- to 8- fold (average 5-fold), compared to non-salp locations, exporting up to 46% of primary production out of the euphotic zone. BCP efficiency increases from 5 to 28% in salp areas, which is among the highest recorded in the global ocean.

Using Chlorophyll Fluorescence to Determine the Fate of Photons Absorbed by Phytoplankton in the World's Oceans.

Approximately 45% of the photosynthetically fixed carbon on Earth occurs in the oceans in phytoplankton, which account for less than 1% of the world's photosynthetic biomass. This amazing empirical observation implies a very high photosynthetic energy conversion efficiency, but how efficiently is the solar energy actually used? The photon energy budget of photosynthesis can be divided into three terms: the quantum yields of photochemistry, fluorescence, and heat. Measuring two of these three processes closes the energy budget. The development of ultrasensitive, seagoing chlorophyll variable fluorescence and picosecond fluorescence lifetime instruments has allowed independent closure on the first two terms. With this closure, we can understand how phytoplankton respond to nutrient supplies on timescales of hours to months and, over longer timescales, to changes in climate.

Ultrafast Energy Transfer Determines the Formation of Fluorescence in DOM and Humic Substances.

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.

Saturation of thylakoid-associated fatty acids facilitates bioenergetic coupling in a marine diatom allowing for thermal acclimation.

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.

Photosynthetic energy conversion efficiency in the West Antarctic Peninsula.

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.

Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum.

Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms.

Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum.

A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

What limits photosynthetic energy conversion efficiency in nature? Lessons from the oceans.

Constraining photosynthetic energy conversion efficiency in nature is challenging. In principle, two yield measurements must be made simultaneously: photochemistry, fluorescence and/or thermal dissipation. We constructed two different, extremely sensitive and precise active fluorometers: one measures the quantum yield of photochemistry from changes in variable fluorescence, the other measures fluorescence lifetimes in the picosecond time domain. By deploying the pair of instruments on eight transoceanic cruises over six years, we obtained over 200 000 measurements of fluorescence yields and lifetimes from surface waters in five ocean basins. Our results revealed that the average quantum yield of photochemistry was approximately 0.35 while the average quantum yield of fluorescence was approximately 0.07. Thus, closure on the energy budget suggests that, on average, approximately 58% of the photons absorbed by phytoplankton in the world oceans are dissipated as heat. This extraordinary inefficiency is associated with the paucity of nutrients in the upper ocean, especially dissolved inorganic nitrogen and iron. Our results strongly suggest that, in nature, most of the time, most of the phytoplankton community operates at approximately half of its maximal photosynthetic energy conversion efficiency because nutrients limit the synthesis or function of essential components in the photosynthetic apparatus.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria.

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.

Phytoplankton. The fate of photons absorbed by phytoplankton in the global ocean.

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.

Energy dissipation pathways in Photosystem 2 of the diatom, Phaeodactylum tricornutum, under high-light conditions.

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.

An RNA interference knock-down of nitrate reductase enhances lipid biosynthesis in the diatom Phaeodactylum tricornutum.

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.

Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light availability.

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.

Studying photoprotective processes in the green alga Chlorella pyrenoidosa using nonlinear laser fluorimetry.

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.

A kinetic model of non-photochemical quenching in cyanobacteria.

High light poses a threat to oxygenic photosynthetic organisms. Similar to eukaryotes, cyanobacteria evolved a photoprotective mechanism, non-photochemical quenching (NPQ), which dissipates excess absorbed energy as heat. An orange carotenoid protein (OCP) has been implicated as a blue-green light sensor that induces NPQ in cyanobacteria. Discovered in vitro, this process involves a light-induced transformation of the OCP from its dark, orange form (OCP(o)) to a red, active form, however, the mechanisms of NPQ in vivo remain largely unknown. Here we show that the formation of the quenching state in vivo is a multistep process that involves both photoinduced and dark reactions. Our kinetic analysis of the NPQ process reveals that the light induced conversion of OCP(o) to a quenching state (OCP(q)) proceeds via an intermediate, non-quenching state (OCP(i)), and this reaction sequence can be described by a three-state kinetic model. The conversion of OCP(o) to OCP(i) is a photoinduced process with the effective absorption cross section of 4.5 × 10(-3)Ų at 470 nm. The transition from OCP(i) to OCP(q) is a dark reaction, with the first order rate constant of approximately 0.1s(-1) at 25°C and the activation energy of 21 kcal/mol. These characteristics suggest that the reaction rate may be limited by cis-trans proline isomerization of Gln224-Pro225 or Pro225-Pro226, located at a loop near the carotenoid. NPQ decreases the functional absorption cross-section of Photosystem II, suggesting that formation of the quenched centers reduces the flux of absorbed energy from phycobilisomes to the reaction centers by approximately 50%.

Photosynthetic energy storage efficiency in Chlamydomonas reinhardtii, based on microsecond photoacoustics.

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.

Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals.

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.

Photoelectron generation by photosystem II core complexes tethered to gold surfaces.

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.

Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro.

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.