Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium.

In the modern ocean, a significant amount of nitrogen fixation is attributed to filamentous, nonheterocystous cyanobacteria of the genus Trichodesmium. In these organisms, nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. Nitrogenase, the enzyme responsible for biological N2 fixation, is irreversibly inhibited by oxygen in vitro. How nitrogenase is protected from damage by photosynthetically produced O2 was once an enigma. Using fast repetition rate fluorometry and fluorescence kinetic microscopy, we show that there is both temporal and spatial segregation of N2 fixation and photosynthesis within the photoperiod. Linear photosynthetic electron transport protects nitrogenase by reducing photosynthetically evolved O2 in photosystem I (PSI). We postulate that in the early evolutionary phase of oxygenic photosynthesis, nitrogenase served as an electron acceptor for anaerobic heterotrophic metabolism and that PSI was favored by selection because it provided a micro-anaerobic environment for N2 fixation in cyanobacteria.

Contribution of aerobic photoheterotrophic bacteria to the carbon cycle in the ocean.

The vertical distribution of bacteriochlorophyll a, the numbers of infrared fluorescent cells, and the variable fluorescence signal at 880 nanometers wavelength, all indicate that photosynthetically competent anoxygenic phototrophic bacteria are abundant in the upper open ocean and comprise at least 11% of the total microbial community. These organisms are facultative photoheterotrophs, metabolizing organic carbon when available, but are capable of photosynthetic light utilization when organic carbon is scarce. They are globally distributed in the euphotic zone and represent a hitherto unrecognized component of the marine microbial community that appears to be critical to the cycling of both organic and inorganic carbon in the ocean.

Bacterial photosynthesis in surface waters of the open ocean.

The oxidation of the global ocean by cyanobacterial oxygenic photosynthesis, about 2,100 Myr ago, is presumed to have limited anoxygenic bacterial photosynthesis to oceanic regions that are both anoxic and illuminated. The discovery of oxygen-requiring photosynthetic bacteria about 20 years ago changed this notion, indicating that anoxygenic bacterial photosynthesis could persist under oxidizing conditions. However, the distribution of aerobic photosynthetic bacteria in the world oceans, their photosynthetic competence and their relationship to oxygenic photoautotrophs on global scales are unknown. Here we report the first biophysical evidence demonstrating that aerobic bacterial photosynthesis is widespread in tropical surface waters of the eastern Pacific Ocean and in temperate coastal waters of the northwestern Atlantic. Our results indicate that these organisms account for 2-5% of the photosynthetic electron transport in the upper ocean.

Cyclic electron flow around Photosystem II in vivo.

The oxygen flash yield (YO2) and photochemical yield of PS II (ΦPS II) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, YO2 and ΦPS II followed each other, suggesting a close coupling between the oxidation of water and QA reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432-443). Following a light-to-dark transition, however, the relationship between QA reduction and the fraction of PS II reaction centers capable of evolving O2 became temporarily uncoupled. ΦPS II recovered to the preillumination levels within 5-10 s, while the YO2 required up to 60 s to recover under aerobic conditions. The recovery of YO2 was independent of the redox state of QA, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (σPS II). The hysteresis between YO2 and the reduction of QA during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.

Effects of Iron Limitation on Photosystem II Composition and Light Utilization in Dunaliella tertiolecta.

The effects of iron limitation on photosystem II (PSII) composition and photochemical energy conversion efficiency were studied in the unicellular chlorophyte alga Dunaliella tertiolecta. The quantum yield of photochemistry in PSII, inferred from changes in variable fluorescence normalized to the maximum fluorescence yield, was markedly lower in iron-limited cells and increased 3-fold within 20 h following the addition of iron. The decrease in the quantum yield of photochemistry was correlated with increased fluorescence emission from the antenna. In iron-limited cells, flash intensity saturation profiles of variable fluorescence closely followed a cumulative one-hit Poisson model, suggesting that PSII reaction centers are energetically isolated, whereas in iron-replete cells, the slope of the profile was steeper and the calculated probability of energy transfer between reaction centers increased to >0.6. Immunoassays revealed that in iron-limited cells the reaction center proteins, D1, CP43, and CP47, were markedly reduced relative to the peripheral light-harvesting Chl-protein complex of PSII, whereas the [alpha] subunit of cytochrome b559 was about 10-fold higher. Spectroscopic analysis established that the cytochrome b559 peptide did not contain an associated functional heme. We conclude that the photochemical conversion of absorbed excitation energy in iron-limited cells is limited by the number of photochemical traps per unit antenna.

Inhibition of PS II photochemistry by PAR and UV radiation in natural phytoplankton communities.

The effects of PAR and UV radiation on PS II photochemistry were examined in natural phytoplankton communities from coastal waters off Rhode Island (USA) and the subtropical Pacific. The photochemical energy conversion efficiency, the functional absorption cross section and the kinetics of electron transfer on the acceptor side of PS II were derived from variable fluorescence parameters using both pump and probe and fast repetition rate techniques. In both environments, the natural phytoplankton communities displayed marked decreases in PS II photochemical energy conversion efficiency that were correlated with increased PAR. In the coastal waters, the changes in photochemical energy conversion efficiency were not statistically different for samples treated with supplementary UV-B radiation or screened to exclude ambient UV-B. Moreover, no significant light-dependent changes in the functional absorption cross section of PS II were observed. The rate of electron transfer between QA (-) and QB was, however, slightly reduced in photodamaged cells, indicative of damage on the acceptor side. In the subtropical Pacific, the decrease in photochemical energy conversion efficiency was significantly greater for samples exposed to natural levels of UV-A and/or UV-B compared with those exposed to PAR alone. The cells displayed large diurnal changes in the functional absorption cross section of PS II, indicative of non-photochemical quenching in the antenna. The changes in the functional absorption cross section were highly correlated with PAR but independent of UV radiation. The time course of changes in photochemical efficiency reveals that the photoinhibited reaction centers rapidly recover (within an hour or two) to their preillumination values. Thus, while we found definitive evidence for photoinhibition of PS II photochemistry in both coastal and open ocean phytoplankton communities, we did not find any effect of UV-B on the former, but a clear effect on the latter. The results of this study indicate that the effects of UV-B radiation on phytoplankton photosynthesis are as dependent on the radiative transfer properties of the water body and the mixing rate, as on the wavelength and energy distribution of the radiation and the absorption cross sections of the biophysical targets.

Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom.

The diadinoxanthin cycle (DD-cycle) in chromophyte algae involves the interconversion of two carotenoids, diadinoxanthin (DD) and diatoxanthin (DT). We investigated the kinetics of light-induced DD-cycling in the marine diatom Phaeodactylum tricornutum and its role in dissipating excess excitation energy in PS II. Within 15 min following an increase in irradiance, DT increased and was accompanied by a stoichiometric decrease in DD. This reaction was completely blocked by dithiothreitol (DTT). A second, time-dependent, increase in DT was detected ∼ 20 min after the light shift without a concomitant decrease in DD. DT accumulation from both processes was correlated with increases in non-photochemical quenching of chlorophyll fluorescence. Stern-Volmer analyses suggests that changes in non-photochemical quenching resulted from changes in thermal dissipation in the PS II antenna and in the reaction center. The increase in non-photochemical quenching was correlated with a small decrease in the effective absorption cross section of PS II. Model calculations suggest however that the changes in cross section are not sufficiently large to significantly reduce multiple excitation of the reaction center within the turnover time of steady-state photosynthetic electron transport at light saturation. In DTT poisoned cells, the change in non-photochemical quenching appears to result from energy dissipation in the reaction center and was associated with decreased photochemical efficiency. D1 protein degradation was slightly higher in samples poisoned with DTT than in control samples. These results suggest that while DD-cycling may dynamically alter the photosynthesis-irradiance response curve, it offers limited protection against photodamage of PS II reaction centers at irradiance levels sufficient to saturate steady-state photosynthesis.

Iron-induced changes in light harvesting and photochemical energy conversion processes in eukaryotic marine algae.

The role of iron in regulating light harvesting and photochemical energy conversion processes was examined in the marine unicellular chlorophyte Dunaliella tertiolecta and the marine diatom Phaeodactylum tricornutum. In both species, iron limitation led to a reduction in cellular chlorophyll concentrations, but an increase in the in vivo, chlorophyll-specific, optical absorption cross-sections. Moreover, the absorption cross-section of photosystem II, a measure of the photon target area of the traps, was higher in iron-limited cells and decreased rapidly following iron addition. Iron-limited cells exhibited reduced variable/maximum fluorescence ratios and a reduced fluorescence per unit absorption at all wave-lengths between 400 and 575 nm. Following iron addition, variable/maximum fluorescence ratios increased rapidly, reaching 90% of the maximum within 18 to 25 h. Thus, although more light was absorbed per unit of chlorophyll, iron limitation reduced the transfer efficiency of excitation energy in photosystem II. The half-time for the oxidation of primary electron acceptor of photosystem II, calculated from the kinetics of decay of variable maximum fluorescence, increased 2-fold under iron limitation. Quantitative analysis of western blots revealed that cytochrome f and subunit IV (the plastoquinone-docking protein) of the cytochrome b(6)/f complex were also significantly reduced by lack of iron; recovery from iron limitation was completely inhibited by either cycloheximide or chloramphenicol. The recovery of maximum photosynthetic energy conversion efficiency occurs in three stages: (a) a rapid (3-5 h) increase in electron transfer rates on the acceptor side of photosystem II correlated with de novo synthesis of the cytochrome b(6)/f complex; (b) an increase (10-15 h) in the quantum efficiency correlated with an increase in D1 accumulation; and (c) a slow (>18 h) increase in chlorophyll levels accompanied by an increase in the efficiency of energy transfer from the light-harvesting chlorophyll proteins to the reaction centers.

Natural Versus Anthropogenic Factors Affecting Low-Level Cloud Albedo over the North Atlantic.

Cloud albedo plays a key role in regulating Earth's climate. Cloud albedo depends on column-integrated liquid water content and the density of cloud condensation nuclei, which consists primarily of submicrometer-sized aerosol sulfate particles. A comparison of two independent satellite data sets suggests that, although anthropogenic sulfate emissions may enhance cloud albedo immediately adjacent to the east coast of the United States, over the central North Atlantic Ocean the variability in albedo can be largely accounted for by natural marine and atmospheric processes that probably have remained relatively constant since the beginning of the industrial revolution.

Effects of Growth Irradiance and Nitrogen Limitation on Photosynthetic Energy Conversion in Photosystem II.

Photosynthetic energy conversion was investigated in five species of marine unicellular algae, (Dunaliella tertiolecta, Thalassiosira pseudonana, T. weisflogii, Skeletorema costatum, Isochrysis galbana) representing three phylogenetic classes, which were grown under steady state conditions with either light or inorganic nitrogen as a limiting factor. Using a pump and probe fluorescence technique we measured the maximum change in variable fluorescence yields, the flash intensity saturation curves for the change in fluorescence yields and the kinetics of the decay in fluorescence yields. Under all growth irradiance levels nutrient replete cells exhibited approximately the same changes in fluorescence yields and similar fluorescence decay kinetics. The apparent relative absorption cross-section of photosystem II, calculated from the slope of the flash intensity saturation curves, generally increased as cells shade adapted. The decay kinetics of the fluorescence yield following a saturating pump flash can be expressed as the sum of three exponential components, with half-times of 160 and 600 microseconds and 30 to 300 milliseconds. The relative contribution of each component did not change significantly with growth irradiance. As cells became more nitrogen limited, however, the maximum change in fluorescence yield decreased, and was accompanied by a decrease in the proportion of a 160 microsecond fluorescence decay component, which corresponds to the transfer of electrons from Q(a) (-) to Q(b). Changes in fluorescence yields were also accompanied by changes in the levels of D1, a protein which is integral in reaction center II, and CP47, a chlorophyll protein forming part of the core of photosystem II. These results are consistent with a loss of functional photosystem II reaction centers. Moreover, in spite of losses of total cellular chlorophyll, which invariably accompanied nitrogen limitation, the apparent absorption cross-sections of photosystem II increased. Our results suggest that nitrogen limitation leads to substantial decreases in photosynthetic energy conversion efficiency.

Comparison of approaches to the instrumental response function in fluorescence decay measurements.

Deconvolution of pulse fluorometry data requires knowledge of the instrumental response, which is not directly observable in some circumstances. Various procedures for approaching the instrumental response function were evaluated for nanosecond fluorescence decay data analyzed by nonlinear least squares, including the commonly used time shift correction and several reference fluorophore methods. A new reference fluorophore technique using a Monte Carlo convolution is introduced and tested. The correction for scattered light in several reference techniques is also presented. The random convolution and one other reference fluorophore method consistently gave superior results over a wide range of experimental conditions.