Submitted to Limnology and Oceanography as a NOTE



Submitted to Limnology and Oceanography as a NOTE DRAFT June 17, 1999

Photosynthetic parameters in the benthos measured in situ

with a Scuba-based fast repetition rate fluorometer

Maxim Y. Gorbunov, Paul G. Falkowski and Zbigniew S. Kolber

gorbunov@ahab.rutgers.edu; falko@imcs.rutgers.edu; zkolber@ahab.rutgers.edu

Environmental Biophysics and Molecular Ecology Program

Institute of Marine and Coastal Sciences

Rutgers, The State University of New Jersey

71 Dudley Road

New Brunswick, New Jersey 08901

Acknowledgements

This research was supported by the Office of Naval Research under Grant # 97PR00617-00. We would like to thank Zvy Dubinsky, Michael Lesser, and Charlie Mazel for helpful suggestions on the instrument design, Eli Perel, Kevin Wyman, Steve Boose, Val Myrnyi, Peter Nawrot for technical assistance, Mike Behrenfeld for helpful comments on the manuscript, and the staff of Caribbean Marine Research Center at Lee Stocking Island for support during field campaigns.

Abstract

We developed a diver-operated Fast Repetition Rate (FRR) Fluorometer for in situ measurements of photosynthetic performance and fluorescence signals in corals, sea grasses, macroalgae, and algal turfs. Due to high rates of primary production, benthic Benthic photoautotrophic organisms can significantly contribute both significantly to the productivity of shallow tropical coastal ecosystems. They also contribute to as well as the remote sensing reflectance signal radiatively transferred from shallow waters. Quantitative assessment of the primary production and the photosynthetic light utilization/dissipation in benthic targets is complicated however, by difficult because of a high level of taxonomic heterogeneitydiversity, spatial heterogeneity, and natural variability in the local nutrient, irradiance, and temperature regimes. To help overcome these problems, we developed a diver-operated Fast Repetition Rate (FRR) Fluorometer for in situ measurements of photosynthetic performance and fluorescence signals in corals, sea grasses, macroalgae, and algal turfs. Using the SCUBA - FRR fluorometer, these processes can be measured in situ with high spatial and temporal resolution. Here we describe We used the instrument design and characteristics and present representative field results for the photosynthetic performance in various benthic organisms in a tropical ecosystem., and to study their photosynthetic response to changes in environmental factors such as nutrient availability and varying light regime.

Benthic photoautotrophic marine organisms, including corals, sea grasses, macroalgae, and algal turfs are among the most productive photosynthetic organisms in aquatic ecosystems (Larkum, 1983; Falkowski and Raven, 1997). However, measurements of photophysiological responses generally require destructive sample manipulations and are limited in spatial and temporal coverage. Moreover, systematic sampling of each of the species comprising these local communities is impractical, or impossible in some protected areas. To overcome these obstacles, we developed a SCUBA-based FRR Fluorometer with an autofocussing imaging system. The instrument permits measurements of an extensive suite of photosynthetic parameters based on fluorescence transients induced by a sequence of brief sub-saturating flashes (Kolber et al., 1998). Here we describe the concept of the diver-operated SCUBA-FRR instrument and present field data that characterizes photosynthetic performance of selected classes of benthic organisms.

Instrument description - The SCUBA-based FRR instrument (Fig. 1) uses a bank of 80 high luminosity blue light-emitting diodes (LED NLPB300, Nichia Chemical Industries, Japan) to excite chlorophyll fluorescence at 460 nm with 30 nm bandwidth. Excitation light is focused on the target using a Fresnel lense (focal length 5 cm), with a spot size of 15 mm. A computer-controlled LED driver circuitry generates a sequence of flashlets with a pulse duration controlled from 0.5 µs to 2 µs, and interval of 2.5 µs to 1 ms. Operating at a pulsed current of 300 mA per LED, the instrument generates about 0.7 W/cm2 of optical power density at a distance of 3 cm from the output window.

The fluorescence signal is collected from a central, 6 mm diameter, portion of the illuminated target, isolated by a red cut-off filter (Schott RG665) and an interference filter (Corion S10-680-R), and detected by an avalanche photodiode module (Hamamatsu C5460). A small portion of the excitation light is recorded by a PIN photodiode as a reference signal. Both the fluorescence and reference signals are integrated over a single excitation pulse by dual switched integrators (Burr-Brown ACF2101) and digitized by 12-bit analog-to-digital converters (LTC1410, Linear Technology). By using an avalanche photodiode as a detector and integrating the fluorescence signal in the analog mode, the instrument operates with a high signal-to-noise ratio, allowing the acquisition of fluorescence transients in a single excitation sequence (see Fig.2), a criterion of crucial importance for diving operations.

The excitation protocols and data acquisition are controlled by an embedded Digital Signal Processing circuitry based on an ADSP-2181 microprocessor (Analog Devices), interfaced to a PC/104 computer board (486DX4 100 MHz, Ampro Computers). Up to 2,000 fluorescence transients can be stored in the on-board Flash Memory Card (SanDisk PCMCIA Type II, 40 Mb). A compact black and white video camera (Marshall Electronics) is incorporated into the instrument, allowing the diver to monitor the target in real-time. A frame grabber (CX-100-30, Imagenation Vision System Specialists) captures the image simultaneously with the fluorescence measurements. A LCD screen installed on the front panel displays both the video signal from the CCD camera and the RGB signal from the on-board PC. A pair of orthogonal, off-axis IR laser diodes (wavelength 780 nm, optical power 5 mW) is incorporated in the viewfinder to precisely control the distance to target. Using an underwater keyboard comprising twelve piezo-keys (Tschudin & Heid Inc., #7020), a diver manipulates the instrument and annotates the acquired data. Photosynthetically available radiation (PAR), temperature, and depth are simultaneously measured by a LiCor 2π underwater quantum meter, a thermistor, and a pressure gauge, all incorporated within the instrument.

Results - SCUBA-FRR fluorometers were used during the Coastal Benthic Optical Properties (CoBOP) field program at Lee Stocking Island, in the Bahamas, during May 1998 and 1999 and January 1999 to study a variety of benthic targets, including corals, seagrass, algal turfs and sediments. Over two thousand measurements were obtained both in situ and in laboratory sea tables. Typical FRR fluorescence profiles measured on zooxanthellate corals, Montastraea cavernosa the seagrass, Thallasia testudinum, and surficial sediment are shown in Fig. 2. The corresponding photosynthetic parameters calculated using the procedure described in (Kolber et al., 1998) are presented in Table 1. These results indicate that different benthic photoautotrophs have characteristic photosynthetic signatures. Zooxanthellae, the symbiotic dinoflagellates algae living in coelenterate coral hosts, are characterized by moderate functional absorption cross-sections, but low quantum yields of photochemistry (Fv/Fm = 0.39). The Algal algal turf, comprising in this region of cyanobacteria, dinoflagellates and diatoms, have the highest σPSII and high photochemical activity (Fv/Fm = 0.50). Finally, seagrasses have small functional cross-sections and very high quantum yields of photochemistry in PSII (Fv/Fm up to 0.70 - 0.73). Such a high level of variability in photosynthetic parameters among benthic objects reflect differences in the molecular structures of the photosynthetic apparatus, as well as in a higher level of morphology.

FRR fluorometry permitted us to explore environmental factors that potentially affect the rates of photosynthetic energy conversion (Kolber et al., 1990; Falkowski and Kolber, 1995; Kolber et al., 1993, 1994; Behrenfeld et al., 1999), such as nutrient availability and ambient light. Nutrient availability and ambient irradiance are the major natural factors directly controlling photosynthetic activity in the ocean (Falkowski and Kolber, 1995). Under nutrient-replete conditions the Fv/Fm ratio averages at 0.65 in wide variety of phytoplankton species, independent of growth irradiance (Falkowski and Kolber, 1995). Nutrient (e.g. nitrogen or iron) limitation leads to a characteristic decline in Fv/Fm, accompanied by a rise in the Fo fluorescence level (Kolber et al., 1988). In zooxanthellae isolated from corals and cultivated on nutrient-replete media, we observed a range of Fv/Fm value from 0.62 to 0.66, similar tonutrient-replete phytoplankton (Kolber et al., 1988). Measurements in a wide variety of zooranthellate corals species, however, revealed an average Fv/Fm of 0.39 ± 0.07 (n = 350), well below the level characteristic for nutrient-replete cells. The low efficiency of photochemical energy conversion in PSII is likely due to nitrogen deficiency of zooxanthellae in hospitace (Falkowski et al., 1993). In contrast to symbiotic zooxanthellae, seagrasses display quantum yields of photochemistry in PSII in a range of 0.70 ± 0.04, suggesting an absence of nutrient limitation. The highest level of variability in the quantum yields of photochemistry in PSII was observed in macroalgae: Fv/Fm ratio varied from 0.50 up to 0.75, reflecting a high level of variability in nutrient status of macroalgae in benthic environment.

Light represents another environmental factor directly controlling photosynthetic efficiency in natural ecosystems. Variations in ambient irradiance induce complex photoadaptive responses in a photosynthetic apparatus, directed to optimize light utilization at low irradiances, while limiting the adverse effects of overexcitation at high irradiances (Baker, and Bowyer, 1994; Falkowski & and Raven, 1997). These processes can be quantified using FRR fluorescence. Fig. 3A shows FRR fluorescence profiles measured on the zooxanthellate coral M. faveolata at various levels of ambient irradiance. Corresponding light-induced changes in the photosynthetic parameters are presented in Fig. 3B. As the ambient light intensity increased, both actual (F’) and maximum (Fm’) fluorescence yields decreased due to non-photochemical quenching of chlorophyll fluorescence (“’” the prime character indicates the measurements are made under ambient light). The variable fluorescence component (ΔF’ = Fm’ - F’) also declined due to combined influence of photochemical and non-photochemical quenching, reducing the quantum yield of photochemistry in PSII (ΔF’/Fm’). The non-photochemical quenching, representing thermal deactivation of the absorbed excitation energy in the light-harvesting antennae, also led to a reduction in σPSII. Under supra-optimal irradiance, when photosynthesis is saturated, photoinhibition of PSII may have also contributed to the light-induced decline in the fluorescence yields. These observations, together with our in situ measurements suggest that under bright light (/ 1000 μmol quanta m-2 s-1), e.g. in shallow waters, up to 20-40 % of the reaction centers of PSII in symbiotic corals may be transiently down regulated.

Fluorescence-derived parameters measured as a function of irradiance allow calculation of the rates of photosynthetic electron transport and reconstruction of photosynthesis versus irradiance (P-E) curves (Kolber and Falkowski, 1993). We express the rate of electron transport through PSII as follows:

Pf = E σPSII ΔF’/Fm’ (1)

Fitting the Pf versus E curve (Fig. 3B) with a model dependence Pf = f(E, α, Pmax) permits retrieval of important photosynthetic parameters, namely, α, the initial slope of the Pf-E curve, and Pmax, the maximum rate of electron transport (Webb et al., 1974; Jassby and Platt, 1976; Platt et al., 1980; Falkowski and Raven, 1997). The light-saturation parameter, Ek, which represents an optimal irradiance on the P-E curve, is defined as:

Ek = Pmax / α (2)

The maximum turnover rate of photosynthetic electron transport (1/τ) can be assessed as the product (Falkowski, 1992):

1/τ = Ek × σPSII (3)

As an example, using the FRR fluorescent data presented in Fig. 3, the following values of the photosynthetic parameters were calculated for PSII reaction centers for a branch of M. faveolata : α = 135 Å2 e quanta-1, Pmax = 115 electrons s-1, Ek = 140 μmol quanta m-2 s-1, and 1/τ = 240 s-1. For a leaf of the seagrass Thallasia T. testudinum the following values were obtained: α = 152 Å2 e quanta-1, Pmax = 415 electrons s-1, Ek = 475 μmol quanta m-2 s-1, and 1/τ = 530 s-1.

Measuring a comprehensive suite of photosynthetic parameters, SCUBA-FRR fluorometry can be used for to monitoring the physiological status of coral reefs in situ and for studying the dynamics of reef response to environmental changes. The precision and robustness of fluorescent measurements achieved in the instrument allows one to follow minute variations in photosynthetic parameters processes in real-time, making this technology an efficient diagnostic tool for monitoring the physiological status of coral reefs and other benthic environments. By analogy with active fluorescent techniques applied to phytoplankton and terrestrial vegetation, we anticipate that the SCUBA-FRR technology will permit an early indication of harmful modifications or stresses to benthic photosynthetic organisms, prior to the appearance of macroscopic changes in the organisms. For example, the instrument can quantify changes in photosynthetic performance of zooxanthellate corals and other benthic organisms in response to such phenomena as temperature induced coral bleaching, eutrophications, anthropogenic pollution, UV exposure, etc. Additionally, understanding the patterns of diel variability of fluorescence-derived photosynthetic parameters provides a basis for modeling daily primary production in coastal environment.

REFERENCES

Baker, N.R. and Bowyer, J.R. (1994) Photoinhibition of photosynthesis from molecular

mechanisms to the field. BIOS Scientific Publishers Ltd., Oxford.

Behrenfeld, M.J. and Kolber, Z. S. (1999) Widespread iron limitation of phytoplankton in the

South Pacific ocean. Science, 283:840-843

Falkowski, P.G., Dubinsky, Z., Muscatine, L., McCloskey, L. (1993) Population control in

symbiotic corals. BioSci. 43: 606-611

Falkowski, P. G. and Kolber, Z. S. (1995) Variations in chlorophyll fluorescence yields in phytoplankton in the world oceans. Aust. J. Plant Physiol. 22: 341-355

Falkowski and Raven (1997) Aquatic photosynthesis, pp. 375. Blackwell Scientific Publishers,

Oxford

Jassby, A.D. and Platt, T. (1976) Mathematical formulation of the relationship between photosynthesis and light for phytoplankton. Limnol. Oceangr. 21:540-547

Kolber, Z., Zehr, J., Falkowski, P.G. (1988) Effects of growth irradiance and nitrogen limitation

on photosynthetic energy conversion in Photosystem II. Plant Physiol 88: 72-79

Kolber, Z. and Falkowski, P.G. (1993) Use of active fluorescence to estimate phytoplankton

photosynthesis in situ. Limnol. and Oceanogr. 38: 1646-1665

Kolber, Z. S., Barber, R. T., Coale, K. H., Fitzwater, S. E., Greene, R. M., Johnson, K. S.,

Lindley, S., Falkowski, P. G. (1994) Iron limitation of phytoplankton photosynthesis in the equatorial Pacific Ocean. Nature 371: 145-149

Kolber, Z., Prasil, O., and Falkowski, P.G. (1998) Measurements of variable chlorophyll

fluorescence using fast repetition rate techniques: defining methodology and

experimental protocols. Biochem Biophys Acta 13 67: 88-106

Larkum, A.W.D. (1983) The primary productivity of plant communities on coral reefs, In: Perspectives on Coral Reefs, D.J. Barnes ed., B.Clouston, Australia.

Platt, T., Gallegos, C.L., and Harrison, W.G. (1980) Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J. Mar. Res. 38:687-701

Webb, W.L., Newton, M., and Starr D. (1974) Carbon dioxide exchange of Alnus rubra: a mathematical model. Oecologica 17:281-291

Table 1. Symbols and abbreviations used throughout the text.

FRR Fast Repetition Rate (fluorometry)

PSII Photosystem II

σPSII functional absorption cross section for PSII (Å2)

Fo, Fm Minimum and maximum yields of chlorophyll-a fluorescence measured after dark adaptation (relative units)

Fv Variable fluorescence ( = Fm - Fo);

Fv/Fm Maximum quantum yield of photochemistry in PSII, dimensionless

p “Connectivity factor” defining the exciton energy transfer between individual photosynthetic units, dimensionless

τQa time constant for photosynthetic electron transport on the acceptor side of PSII (Qa reoxidation) (s)

F’, Fm’ Actual and maximum yields of chlorophyll-a fluorescence measured at ambient light (relative units)

ΔF’/Fm’ Quantum yield of photochemistry in PSII measured under ambient light

E Irradiance (μmol quanta m-2 s-1)

Pf Rate of photosynthetic electron transport through PSII (e s-1)

Pmax Maximum rate of electron transport (e s-1)

← Initial slope of the Pf-E curve (Å2 e quanta-1)

Ek Light-saturation parameter (μmol quanta m-2 s-1)

1/τ Maximum turnover rate of photosynthesis (s-1)

Table 2. Fluorescent and photosynthetic parameters calculated from the FRR profiles presented in Fig.2.

| |Fv/Fm |σPSII (Å2) |Fm (a.u.) |p |τQa (μs) |

|Coral |0.38 ± 0.01 |410 ± 20 |610 ± 5 |0.15 |530 ± 100 |

|Sediment |0.55 ± 0.01 |530 ± 20 |350 ± 2 |0.30 |475 ± 80 |

|Seagrass |0.73 ± 0.01 |190 ± 6 |455 ± 5 |0.55 |560 ± 50 |

Figure captures.

Figure 1. Block diagram of the SCUBA-based Fast Repetition Rate Fluorometer.

Figure 2. Chlorophyll fluorescence transients measured with the FRR protocol on the zooxantellate coral Montastraea cavernosa, the seagrass Thallasia testudinum and algal turf on sediment. On the first phase (saturation protocol) a series of 64 sub-saturating flashlets of 1.5 μs duration was used to cumulatively saturate PSII within 150 μs. A magnitude of the rise in fluorescence yield is determined by the quantum yield of photochemistry in PSII (Fv/Fm), while the rate of the fluorescence rise is proportional to the functional absorption cross section of PSII (σPSII). Upon cessation of the saturation protocol, the fluorescence yield decreases, reflecting kinetics of electron transfer on the acceptor side of PSII. A series of the weak flashes of 0.5 μs duration at 100 to 800 μs intervals was used to monitor the relaxation kinetics.

Figure 3. (A) FRR fluorescent transients measured in the coral Montastraea faveolata at various levels of ambient light. (B) Light-induced changes in photosynthetic parameters calculated from the FRR profiles.

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