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Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals[para][dagger]
Photochemistry and Photobiology, May 2003 by Gilmore, Adam M, Larkum, Anthony W D, Salih, Anya, Itoh, Shigeru, Et al
ABSTRACT
Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef-building coral tissue. We used a streak-camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (
Abbreviations: CCD, charged coupled device; chl, chlorophyll; DCI, double-convolution integral; FP, fluorescent protein; FRET, Forster resonance energy transfer; LSGRG, large-scale general reduced gradient; PSII, photosystem II; [alpha]([tau]), pre-exponential amplitude factor as a function of the fluorescence lifetime.
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INTRODUCTION
The sun-exposed tissues of reef-building corals may contain both fluorescent proteins (FP), which are related to the well-known green FP of Aequorea victoria, and photosynthetic algal symbionts known as zooxanthellae (1-7). Both the FP and the chlorophyll (chl)-containing algae may absorb visible light and consequently reemit the absorbed light as fluorescence at longer wavelengths. The FP and zooxanthellae density distribution varies considerably among coral genera, coral species and environmental circumstances (4,8-10). Recent concerns over rising global temperatures of coastal waters are associated with observations of environmentally stressed corals that lose their natural coloration because of changes in the content and composition of both zooxanthellae and FP (11-13). This loss of coloration is referred to as "coral bleaching" and is primarily associated with expulsion of the zooxanthellae from the coral tissue along with concomitant decomposition of the chromatophoric FP. Coral bleaching is believed to be closely associated with the deterioration of the physiological state of the zooxanthellae that is itself reflected in their diminished ability to use absorbed light for photosynthesis (8,14,15). The latter is of primary physiological concern because algal photosynthesis provides the primary source of energy for the host coral animal.
The photosynthetic efficiency of the zooxanthellae is routinely measured with techniques involving chl a fluorescence (8,14-17), which provides a nondestructive measure of the primary photochemical efficiency of photosystem II (PSII). A recent report by Salih et al. (8) showed a positive correlation between both FP content and color variation and PSII efficiency of the zooxanthellae under excess light stress. It was shown that photoinhibition of PSII in zooxanthellae due to high light was reduced in highly fluorescent color morphs with dense FP tissue populations compared with zooxanthellae from morphs of the same species lacking FP. These researchers proposed that the primary photoprotective mechanism of the FP for the zooxanthellae and vulnerable coral tissues involved light screening, energy dissipation via fluorescence and scattering of excess visible and UV light (8,18,19). The FP bodies in many cases are spatially arranged in the tissue and key organelles of the animal in such a way as to effectively filter or reflect excess light away from the symbiotic algae.
Studies have also proposed that in contrast to light screening and reflection, in deep waters the FP may act as accessory light-harvesting pigment bodies for the zooxanthellae (4,20,21). This hypothesis was recently discussed in the context of empirical observations from isolated protein and pigment systems that light reemitted by FP exhibits spectral overlap with, and thus exhibits possible energetic favorability for absorption by, the chl and carotenoid molecules in the algae's light-harvesting apparatus (4). The light-harvesting hypothesis further involves the possibility that FP may absorb light in the UV region, which is not absorbed readily by chl, and then reemit light at a longer wavelength (i.e. the emission is Stokes-shifted [22]) that is capable of being harvested by the endosymbiotic algae. In this manner the FP would function to expand the spectral range of photosynthetically active radiation from the normal range of 400-700 nm to shorter wavelengths.
In almost all cases FP comprise heterogeneous pools (8,10), normally of blue, blue-green and green emitters as well as of yellow and red emitters. It was considered that existence of FP mixtures with varying absorption and emission energy levels may function to transfer energy downhill within an FP array by a radiative energy transfer mechanism from bluer to greener or redder emitting-absorbing forms (8,19,23). This radiant energy transfer cascade was hypothesized to facilitate the conversion of damaging high-energy UV wavelengths (
In this study we simultaneously acquired and analyzed picosecond time-resolved fluorescence emission spectra of the FP and zooxanthellae in living, nonstressed coral tissue, including specimens collected from both Okinawa, Japan, and Sydney, Australia. The data were collected with a streak-camera spectrograph that simultaneously records both the time and wavelength dependence of the fluorescence emission, Excitation by a blue laser pulse simultaneously excited both FP and chl in the zooxanthellae photosystems. The data were analyzed using the double-convolution integral (DCI) method (24), which facilitates high-resolution time- and spectral-resolved FRET simulations because it substantially reduces the number of free model parameters compared with conventional decay-associated spectral analysis techniques. The results are discussed in light of the two main observations, namely, (1) the inefficient transfer of energy from the FP to the zooxanthellae chl; and (2) the evidence provided for FRET within the FP populations of all samples.
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