We present a methodology, called fast repetition rate (FRR) fluorescence, that measures the functional absorption cross-section (sigmaPS II) of Photosystem II (PS II), energy transfer between PS II units (p), photochemical and nonphotochemical quenching of chlorophyll fluorescence, and the kinetics of electron transfer on the acceptor side of PS II. The FRR fluorescence technique applies a sequence of subsaturating excitation pulses ('flashlets') at microsecond intervals to induce fluorescence transients. This approach is extremely flexible and allows the generation of both single-turnover (ST) and multiple-turnover (MT) flashes. Using a combination of ST and MT flashes, we investigated the effect of excitation protocols on the measured fluorescence parameters. The maximum fluorescence yield induced by an ST flash applied shortly (10 &mgr;s to 5 ms) following an MT flash increased to a level comparable to that of an MT flash, while the functional absorption cross-section decreased by about 40%. We interpret this phenomenon as evidence that an MT flash induces an increase in the fluorescence-rate constant, concomitant with a decrease in the photosynthetic-rate constant in PS II reaction centers. The simultaneous measurements of sigmaPS II, p, and the kinetics of Q-A reoxidation, which can be derived only from a combination of ST and MT flash fluorescence transients, permits robust characterization of the processes of photosynthetic energy-conversion.
We describe the theory and practice of estimating photosynthetic rates from light-stimulated changes in the quantum yield of chlorophyll fluorescence. By means of a pump-and-probe fluorescence technique, where weak probe flashes are used to measure the change in the quantum yield of fluorescence induced by the strong pump flash, it is possible to derive the absolute absorption cross sections for photosystem 2, the quantum yield for photochemistry, and the maximum rate of photosynthetic electron transport at light saturation. In conjunction with a semiempirical biophysical model of photosynthesis, these parameters can bc used to calculate the instantaneous rate of gross photosynthesis in situ under ambient irradiance. A profiling pump-and-probe fluorometer was constructed and interfaced with a CTD, and vertical profiles of variable fluorescence were obtained on four cruises in the northwest Atlantic Ocean. The derived photosynthetic rates were compared with concurrent estimates of production based on radiocarbon uptake. The correlation coefficient between the two estimates of primary production, normalized to Chl a, was 0.86; linear regression analysis yielded a slope of 1.06. There is a 3-4-fold range in the maximum change in the quantum yields of photochemistry and absorption cross-sections in natural phytoplankton communities. Uncertainties in the pump-and-probe-derived estimates of photosynthesis arc primarily due to temporal mismatches between instantaneous and time-integrated measures of production and in biological variability in the ratio of the number of PS2 reaction centers to total Chl a.
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.
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 N 2 fixation, is irreversibly inhibited by oxygen in vitro. How nitrogenase is protected from damage by photosynthetically produced O 2 was once an enigma. Using fast repetition rate fluorometry and fluorescence kinetic microscopy, we show that there is both temporal and spatial segregation of N 2 fixation and photosynthesis within the photoperiod. Linear photosynthetic electron transport protects nitrogenase by reducing photosynthetically evolved O 2 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 N 2 fixation in cyanobacteria.
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