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

We thus propose, consistent with the suggestions of Dove et al. (4) and Salih et al. (8), that FP absorption in the UV to blue-green regions of the solar emission spectrum most likely serves to intercept, scatter and block excess visible and potentially damaging UV excitation from reaching the symbionts and other vital organelles of the host coral. It was clear and potentially physiologically relevant that the highly fluorescent blue FP morph of P. versipora exhibited higher light absorption, especially in the UV, compared with the less fluorescent green morph. In this light, our recent unpublished results indicate that the overall FP density in the coral tissue is typically considerably higher in the blue as opposed to green color morphs of P. versipora.

Because of the low-integrated intensity (

One final point of interest comparing the fluorescence lifetime distributions of the algal PSII and the FP was the clear differences in the resolved widths of the fluorescence lifetime distributions. As explained in earlier studies (32,34,35), the width of a fluorescence lifetime distribution of a protein-bound fluorophore, like the FP and algal chl, may be interpreted to indicate heterogeneity in the molecular environment of the fluorophore. These ideas are in accordance to the theories of protein conformational substates and dynamics proposed by Frauenfelder (36,37) and related theories regarding the kinetic behavior of protein-bound fluorophores promulgated by Alcala et al. (38,39). Because of the narrowness of the FP fluorescence lifetime distributions in this study, we interpret the main FP emission to emanate from a homogeneous population of fluorophores, probably representing proteins with fluorophores in very similar molecular environments. As explained before, PSII (35) lifetime distributions at room temperature are generally broad (>100 ps in many cases), and the width may be attributed to a variety of heterogeneity factors including the PSII chl-protein environment and dynamic PSII photochemical activities of the sample, inter alia.

Evidence for Forster resonance energy transfer among fluorescent proteins in vivo

It is clear that FP emission observed in all specimens of this study is influenced by minor, rapid spectral shift components that we attribute to FRET. The spectral shifts at room temperature always indicate downhill energy transfer as evidenced by reciprocal amplitudes decreasing in the blue and increasing in the greener wings of the spectrum. We speculate that in each color morph the initial donor species may relate primarily to minor pools of pigments that are likely to be in close spatial association with each other in oligomeric form (40). Perhaps the oligomers are present either in the granular FP bodies or in intracellular FP bodies that are not enclosed in FP granules (8). The donors may relate to the same or similar protein structures as the acceptors with the altered spectral properties perhaps attributable to altered chemical properties (pH) or environments (electrostatic factors) of the amino acid residues associated with the FP fluorophore (3,6), inter alia. The close structural similarity of the donor-acceptor pairs is reasoned because, as previously noted, the main FP emission component is a predictor for the initial peaks resolved in the early phases of the FP excitation.

Moreover, it is established from recent time-resolved fluorescence studies in vitro that oligomers comprising both immature green and mature red forms of the coral FP known as Discosoma Reel (Ds-Red) exhibit FRET (41,42). The reciprocal kinetic responses observed in vitro generally correspond with those obtained here in vivo with the blue and green FP. Notably, in comparison with one of the clearest case studies (41), our main decay component was faster for the blue-green acceptor

CONCLUSIONS

This study demonstrates the use of in vivo simultaneous time and wavelength fluorescence acquisition and DCI analysis (27) for answering important physiological questions, namely, (1) that the blue and green FP do not function in light harvesting for reef-building corals that dwell in shallow water; and (2) that FRET is a primary process in the excited-state behavior of FP in reef-building corals. This study provides a foundation of data from nonstressed corals from key geographical regions of interest with respect to coral bleaching (13,44,45) based on which future studies of stress conditions may be initiated and compared. Future work should be aimed at exploring the mechanism of the proposed photoprotective function of the FP and the physiological state of both the FP and zooxanthellae in response to coral-bleaching episodes. The fluorescence lifetimes and spectra should provide a valid quantitative indicator, especially under conditions where the tissue concentrations and chemical properties of either zooxanthellae or FP are likely to change simultaneously. This is because determination of the fluorescence intensities and ratios alone is complicated by concomitant changes in the concentration and excited-state lifetimes of fluorophores in the tissue. Finally, the methods used for identification of the new FP and FRET partners described in this study may be of interest to researchers who wish to isolate, clone and mutate FP for molecular technology, as is becoming increasingly common for the green and red FP from other cnidarians.