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Euglena gracilis photoreception interpreted by microspectroscopy
Europ. J. Protistol. 39, 404–409 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp
Euglena gracilis photoreception interpreted by microspectroscopy Vincenzo Passarelli, Laura Barsanti, Valtere Evangelista, Anna Maria Frassanito and Paolo Gualtieri* Istituto di Biofisica, CNR, via Moruzzi 1, 56124 Pisa, Italy; E-mail: [email protected] Received: 4 September 2003; 3 October 2003. Accepted: 4 October 2003
For over 100 years, Euglena gracilis has represented a major subject for photobiological studies. This unicellular flagellate is well suited for such investigations because of its special complement of organelles that may be considered an ancient, yet complete “visual” system. The actual photoreceptive roles of the cytoplasmic eyespot and the photoreceptor (old terminology: paraflagellar swelling) of E. gracilis are still under debate, because of the different experimental evidence produced by the different research groups still working on this organism. This paper presents the analysis of the spectroscopical measurements performed by our group, which forms the basis of a possible model for Euglena photoresponses. Key words: Euglena; Microspectroscopy; Microspectrofluorimetry; Photobehaviour; Photoreception.
Introduction Euglena gracilis is a photosynthetic and photosensitive flagellate that dwells in natural shallow ponds, and uses sunlight as a source of energy and information. Its chloroplasts are the energy-capturing devices, whereas a simple but sophisticated system is used as a light detector. Two flagella are inserted in a subapical invagination of the cell termed the reservoir. The eyespot, composed of red-orange pigment granules, lies in the adjacent cytoplasm. Only one flagellum emerges from the cell and consists of an axoneme, a paraxial rod running parallel to it, and a photoreceptor near its base (Gualtieri et al. 1990; Verni et al. 1992). This configuration of eyespot, photoreceptor and flagellum can be considered a simple but complete visual system, which has made Euglena an intriguing subject for photobiological studies for more than a century. During the revolving motion of the cell along *corresponding author 0932-4739/03/39/04-404 $ 15.00/0
its swimming path, the eyespot comes between the light source and the photoreceptor, thus modulating the light that reaches this organelle, and regulating the steering of the locomotory flagellum. The photoreceptor, a three-dimensional proteic crystal, is composed of a stack of more than 100 crystalline layers with a regular organization of component proteins. The layers, with a height of about 7 nm, seem to be constituted of the same protein (Walne et al. 1998); as a consequence the photoreceptor can be described as an ordered gel with a very low degree of fluidity that could impose radial pressure on the proteins, and decrease the solvatation of their surfaces (Stuart et al. 2003). In this 3D crystal lattice, due to the limited flexibility, the protein or proteins are either forced to assume only a few of the possible conformational states, or a novel conformational structure is actually induced (Gabellieri et al. 1988). Since the photoreceptive protein(s) undergoes a natural three-
Euglena photoreception
dimensional crystallization to form the photoreceptor of Euglena, it must possess a quite peculiar structure, which possibly restricts chromophore movements inside the binding pocket so as to highly influence the spectroscopic properties of the protein (Pesce 1971). Moreover, Euglena photoreceptor proteins behave as all the prokaryotic and eukaryotic light-sensory proteins, i.e. it is capable of a cyclic photoregeneration, with identified intermediates (Knowles and Dartnall 1977; Marwan and Oesterhelt 1990; Bogomolni and Spudich 1991; Barsanti et al. 2000). To provide important information on the series of structural changes that the Euglena photoreceptive protein(s) undergoes in the photoreceptor in response to light, and to suggest a model for the photobehavioural responses of the cell, we here report a summary of the “in vivo” absorption and fluorescence spectra of its photoreceptive apparatus, i.e. the photoreceptor and eyespot.
Materials and methods Cultures Cultures of Euglena gracilis strain Z (Sammlung Von AlgenKulturen Gottingen, 1224-5/25) cells were grown axenically in autotrophic Cramer-Myers medium 0.025 M in sodium acetate (pH 6.8) (Cramer and Myers 1952), under constant temperature (24 °C) and continuous illumination (2 × 102 µmol photons m–2 sec–1).
Absorption and Fluorescence Microscopy and spectrum analysis The apparatus used in our laboratory to perform absorption measurements has been previously described in detail (Gualtieri et al. 1989). A 3 second illumination of the sample with a radiant flux of about 10–10 J enabled us to obtain a spectrum which involved calculation of optical density by averaging 10,000 values for each wavelength, with a smallest reliably measurable optical density value of 3.6 × 10–2. The instrumentation used to obtain fluorescence spectra was a home-made set-up previously described in detail by Evangelista et al. (2003). Fluorescence spectra of the Euglena photoreceptor were acquired with two filter combinations, a UV-blue set and a blue-violet set. These two filter combinations are the only two available due to the spectral distribution in the UV-blue range of the Mercury high-pressure lamp. Spectra decomposition and non-linear fitting were performed by means of Origin 7.0 (Origin Lab, MA, USA) and Mathematica 4.0 software packages (Wolfram Res., IL, USA).
405
Results and discussion The presence in the photoreceptor of Euglena of a photochromic pigment, which undergoes lightdriven reversible photochromism has been well established by means of digital and fluorescence microscopy (Barsanti et al. 1997). The photoreceptor possesses optical bistability, i.e. upon photoexcitation the ground state generates a stable excited state, which can be photochemically driven back to the ground state. The 27 kDa protein extracted from the photoreceptor shows a similar behaviour, the photochromic reaction cycling between two different stable conformers, the parent and the excited conformers (Barsanti et al. 2000). The quantum yield of the forward reaction is higher than that of the reverse reaction, both being close to unity. No thermal de-activation occurs. In order to produce a photo-cycle, a 365 nm excitation light must be used (Fig. 1a). A very low fluorescence signal is present at the beginning (continuous gray line), but upon illumination we can observe a green emission that increases and reaches saturation after 8 seconds (continuous black line). Therefore, after the 8 seconds of 365 nm excitation, the photo-generation of the fluorescent excited state is complete. Gaussian bands decomposition of the emission spectrum reveals 4 bands with different intensity centered at about 480 nm, 504 nm, 525 nm, and 556 nm (dashed lines). Figure 1b shows the fluorescence spectrum of a single photoreceptor under 436 nm excitation light, after 10 seconds of excitation with the 365 nm light. A maximum fluorescence signal arises after the insertion of the blue-violet filter (continuous black line). Upon illumination by 436 nm light we can observe a fading of the green emission that decreases to almost zero after 10 seconds (continuous gray line). Regeneration of the non-fluorescent ground state is almost complete. Gaussian bands decomposition of the emission spectrum reveals 3 bands with different intensity centered at about 500 nm, 525 nm, and 556 nm (dashed lines). The bands obtained by the Gaussian decomposition indicate the presence of a mixture of very similar conformers in the photoreceptor, each one capable of photocycling between a non-fluorescent parent species and a fluorescent excited species. Figure 1c shows a very simple scheme of the photo-cycle of the Euglena photoreceptor: illumination at 365 nm of the non-fluorescent ground state leads to the photo-generation of the fluores-
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V. Passarelli et al.
Fig. 1. a. Fluorescence spectrum of a photoreceptor excited at 365 nm; b. fluorescence spectrum of a photoreceptor excited at 436 nm; c. scheme of the photoreceptor photocycle. In a and b the dashed lines are Gaussian decomposition bands. See text for details.
Euglena photoreception
407
Fig. 2. a. Absorption spectra of the ground (continuous gray line) and excited (continuous dark line) state of the photoreceptor, showing the Gaussian decomposition bands (dotted lines), with superimposed absorption spectrum of the eyespot (dashed line); b. difference spectrum between the absorption spectra of the ground and excited state of the photoreceptor.
cent excited state, which in turn is driven back to the ground state by illumination at 436 nm (Evangelista et al. 2003). Figure 2a shows the absorption spectrum of the ground state (continuous gray line) of the photoreceptor (Gualtieri et al. 1989). Its structure is rather complex: at least two Gaussian bands can be detected in the α band, one centered at 456 nm, with a 15% area, and the other centered at 501 nm, with a 85% area (Fig. 2a, dotted lines). These two bands should correspond to the excited conformers (456 nm) and parent conformers (501 nm) of the
photoreceptive protein present inside the photoreceptor, already shown by the fluorescence spectra. Since we know the two wavelengths which induce
Fig. 3. Scheme of the photoreceptor photocycle showing the screening effect of the eyespot.
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V. Passarelli et al.
the transition from the ground state to the excited state (365 nm) and from the excited back to the ground state (436 nm), we can infer a difference spectrum between these two states. This difference spectrum shows a positive deviation corresponding to the wavelengths that cause the transition of the ground state to the excited state, around 365 nm, and a negative deviation corresponding to the wavelengths that cause the opposite transition, from the excited back to the ground state, around 436 nm. So far, we have not succeeded in measuring the absorption spectrum of the excited state of the photoreceptor, due to difficulties of setting-up instrumentation for measuring this kind of spectrum. However, a possible absorption spectrum of the excited state (Fig. 2a, continuous dark line) can
be obtained by inverting the areas of the two bands corresponding to the two conformer species of the photoreceptive protein, assigning the 85% area to the band centered at 456 nm, and 15% area to the band at 501 nm (Fig. 2a, dotted lines). In the excited state of the photoreceptor the excited protein conformer should be present in higher amount with respect to the parent conformer. Using the hypothetical absorption spectrum of the excited state of the photoreceptor, we obtain a difference spectrum that best fits the inferred difference spectrum: this spectrum possess a positive band centered around 365 nm and a bigger negative band centered around 436 nm (Fig. 2b). Figure 2a also shows the absorption spectrum of the eyespot (dashed line). The analysis of this spec-
Fig. 4. Example of the dynamics of the light alignment of Euglena obtained by mathematical simulation; e = eyespot; p = photoreceptor.
Euglena photoreception
trum shows that it can be superimposed on to the α band of the absorption spectrum of the excited form of the photoreceptor, with the same decomposition bands. We can use all the gathered spectroscopic information to interpret Euglena photo-responses. As the cell rotates while swimming (rotation frequency: 2 Hz; Ascoli et al. 1978), the photoreceptor experiences periodic shading by the eyespot, which comes between the light source and the organelle. This shading was believed to trigger the photophobic responses that regulate cell orientation. The classic interpretation of Euglena photobehaviour by Jennings (1906) considered a change in the intensity of light the determining cause of the cell reaction movements. A century later, our data allow us to improve and refine this model. It is not a change in light intensity that leads to the cell reorientation, but a selective screening of the absorption window of the excited state of the photoreceptor by the eyespot. Since the absorption spectra of the eyespot and that of the excited state of the photoreceptor are super-imposable, the screening effect of the eyespot leads to the interruption of the photo-cycle, preventing the transition of the excited state of the photoreceptor to its ground state (Fig. 3). During all the shading period, the photoreceptor is in the excited state, which probably generates a photoelectric signal due to the change in dipole orientations between the excited and parent conformers of the photoreceptive protein. Under the influence of this electric field, a displacement photocurrent could be propagated through paraxial rod filaments via charge transfer between rod proteins, which in turn generates longitudinal waves of contraction along the paraxial rod. The contractility of the rod should regulate the flagellum movement, turning the cell body toward the light source (Walne et al., 1998). The ground state of the photoreceptor is restored when the photoreceptor again achieves alignment with the light. An example of the dynamics of the light alignment of a model Euglena obtained by resolving two sets of equations, one relating to motion of the cell body and the other to motion of the flagellum (which space does not allow us to elaborate here), is shown in Figure 4. The initial orientation of the cell is opposite to the light direction, but in about 10 s, which correspond to 20 revolutions around
409
the cell main axis, Euglena is completely aligned with the light.
References Ascoli C., Barbi M., Frediani C. and Mure A. (1978): Measurements of Euglena motion parameters by laser light scattering. Biophys. J. 24, 585–599. Barsanti L., Passarelli V., Walne P. L., and Gualtieri P. (1997): In vivo photo-cycle of the Euglena gracilis photoreceptor. Biophys. J. 72, 545–553. Barsanti L., Passarelli V., Walne P. L., and Gualtieri P. (2000): The photoreceptor protein of Euglena gracilis. FEBS Letters 482, 247–251. Bogomolni R. A. and Spudich J. L. (1991): Archaebacterial rhodopsins: sensory and energy transducing membrane proteins. In: Spudich J. L., and Satir B. H. (eds.): Sensory Receptors and Signal Transduction, pp. 233–255, WileyLiss, New York. Cramer M. and Myers J. (1952): Growth and photosynthetic characteristics of Euglena gracilis. Arch. Mikrobiol. 17, 384–402. Evangelista V., Passarelli V., Barsanti L. and Gualtieri P. (2003): Fluorescence behaviour of Euglena photoreceptor. Photochem. Photobiol. 78, 93–97. Gabellieri E., Strambini G. B. and Gualtieri P. (1988): Tryptophan phosphorescence and the conformation of liver alcohol dehydrogenase in solution and in the crystalline state. Biophys. Chem. 30, 61–67. Gualtieri P., Barsanti L. and Passarelli V. (1989): Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis. Biochim. Biophys. Acta 993, 293–296. Gualtieri P., Barsanti L., Passarelli V., Verni F., and Rosati G. (1990): A look into the reservoir of Euglena: SEM investigation of the flagellar apparatus. Micron Microsc. Acta 21, 131–138. Jennings, H. S. (1906): Behavior of the lower organisms. Columbia University Press, New York. Knowles A. and Dartnall H. J. A. (1977): Photochemistry of extracted visual pigments. In: Davson H. (ed.): The eye Vol. 2B, The photobiology of vision pp. 289–345, Academic Press, New York. Marwan W. and Oesterhelt D. (1990): Quantitation of photochromism of sensory rhosopsin-I by computerized tracking of Halobacterium halobium cells. J. Mol. Biol. 215, 277–285. Pesce A. J., Rosen C. and Patsby T. L. (1971): Fluorescence spectroscopy, Marcel Dekker Inc., New York, USA. Stuart, J. A., D. L., Marcy, K. J., Wise and R. R. Birge (2003): Biomolecular electronic device applications of bacteriorhodopsin. In: Barsanti L., Evangelista V., Gualtieri P., Passarelli V. and Vestri S. (eds.): Molecular electronics: biosensors and biocomputers, pp. 265–299, Kluwer, Amsterdam. Verni F., Rosati G., Lenzi P., Barsanti L., Passarelli V., and Gualtieri, P. (1992): Morphological relationship between paraflagellar swelling and paraxial rod in Euglena gracilis. Micron Microsc. Acta 23, 37–44. Walne P. L., Passarelli V., Barsanti L. and Gualtieri, P. (1998): Rhodopsin: a photopigment for phototaxis in Euglena gracilis. Crit. Rev. Plant Sci. 17, 559–574.
Euglena gracilis photoreception interpreted by microspectroscopy Vincenzo Passarelli, Laura Barsanti, Valtere Evangelista, Anna Maria Frassanito and Paolo Gualtieri* Istituto di Biofisica, CNR, via Moruzzi 1, 56124 Pisa, Italy; E-mail: [email protected] Received: 4 September 2003; 3 October 2003. Accepted: 4 October 2003
For over 100 years, Euglena gracilis has represented a major subject for photobiological studies. This unicellular flagellate is well suited for such investigations because of its special complement of organelles that may be considered an ancient, yet complete “visual” system. The actual photoreceptive roles of the cytoplasmic eyespot and the photoreceptor (old terminology: paraflagellar swelling) of E. gracilis are still under debate, because of the different experimental evidence produced by the different research groups still working on this organism. This paper presents the analysis of the spectroscopical measurements performed by our group, which forms the basis of a possible model for Euglena photoresponses. Key words: Euglena; Microspectroscopy; Microspectrofluorimetry; Photobehaviour; Photoreception.
Introduction Euglena gracilis is a photosynthetic and photosensitive flagellate that dwells in natural shallow ponds, and uses sunlight as a source of energy and information. Its chloroplasts are the energy-capturing devices, whereas a simple but sophisticated system is used as a light detector. Two flagella are inserted in a subapical invagination of the cell termed the reservoir. The eyespot, composed of red-orange pigment granules, lies in the adjacent cytoplasm. Only one flagellum emerges from the cell and consists of an axoneme, a paraxial rod running parallel to it, and a photoreceptor near its base (Gualtieri et al. 1990; Verni et al. 1992). This configuration of eyespot, photoreceptor and flagellum can be considered a simple but complete visual system, which has made Euglena an intriguing subject for photobiological studies for more than a century. During the revolving motion of the cell along *corresponding author 0932-4739/03/39/04-404 $ 15.00/0
its swimming path, the eyespot comes between the light source and the photoreceptor, thus modulating the light that reaches this organelle, and regulating the steering of the locomotory flagellum. The photoreceptor, a three-dimensional proteic crystal, is composed of a stack of more than 100 crystalline layers with a regular organization of component proteins. The layers, with a height of about 7 nm, seem to be constituted of the same protein (Walne et al. 1998); as a consequence the photoreceptor can be described as an ordered gel with a very low degree of fluidity that could impose radial pressure on the proteins, and decrease the solvatation of their surfaces (Stuart et al. 2003). In this 3D crystal lattice, due to the limited flexibility, the protein or proteins are either forced to assume only a few of the possible conformational states, or a novel conformational structure is actually induced (Gabellieri et al. 1988). Since the photoreceptive protein(s) undergoes a natural three-
Euglena photoreception
dimensional crystallization to form the photoreceptor of Euglena, it must possess a quite peculiar structure, which possibly restricts chromophore movements inside the binding pocket so as to highly influence the spectroscopic properties of the protein (Pesce 1971). Moreover, Euglena photoreceptor proteins behave as all the prokaryotic and eukaryotic light-sensory proteins, i.e. it is capable of a cyclic photoregeneration, with identified intermediates (Knowles and Dartnall 1977; Marwan and Oesterhelt 1990; Bogomolni and Spudich 1991; Barsanti et al. 2000). To provide important information on the series of structural changes that the Euglena photoreceptive protein(s) undergoes in the photoreceptor in response to light, and to suggest a model for the photobehavioural responses of the cell, we here report a summary of the “in vivo” absorption and fluorescence spectra of its photoreceptive apparatus, i.e. the photoreceptor and eyespot.
Materials and methods Cultures Cultures of Euglena gracilis strain Z (Sammlung Von AlgenKulturen Gottingen, 1224-5/25) cells were grown axenically in autotrophic Cramer-Myers medium 0.025 M in sodium acetate (pH 6.8) (Cramer and Myers 1952), under constant temperature (24 °C) and continuous illumination (2 × 102 µmol photons m–2 sec–1).
Absorption and Fluorescence Microscopy and spectrum analysis The apparatus used in our laboratory to perform absorption measurements has been previously described in detail (Gualtieri et al. 1989). A 3 second illumination of the sample with a radiant flux of about 10–10 J enabled us to obtain a spectrum which involved calculation of optical density by averaging 10,000 values for each wavelength, with a smallest reliably measurable optical density value of 3.6 × 10–2. The instrumentation used to obtain fluorescence spectra was a home-made set-up previously described in detail by Evangelista et al. (2003). Fluorescence spectra of the Euglena photoreceptor were acquired with two filter combinations, a UV-blue set and a blue-violet set. These two filter combinations are the only two available due to the spectral distribution in the UV-blue range of the Mercury high-pressure lamp. Spectra decomposition and non-linear fitting were performed by means of Origin 7.0 (Origin Lab, MA, USA) and Mathematica 4.0 software packages (Wolfram Res., IL, USA).
405
Results and discussion The presence in the photoreceptor of Euglena of a photochromic pigment, which undergoes lightdriven reversible photochromism has been well established by means of digital and fluorescence microscopy (Barsanti et al. 1997). The photoreceptor possesses optical bistability, i.e. upon photoexcitation the ground state generates a stable excited state, which can be photochemically driven back to the ground state. The 27 kDa protein extracted from the photoreceptor shows a similar behaviour, the photochromic reaction cycling between two different stable conformers, the parent and the excited conformers (Barsanti et al. 2000). The quantum yield of the forward reaction is higher than that of the reverse reaction, both being close to unity. No thermal de-activation occurs. In order to produce a photo-cycle, a 365 nm excitation light must be used (Fig. 1a). A very low fluorescence signal is present at the beginning (continuous gray line), but upon illumination we can observe a green emission that increases and reaches saturation after 8 seconds (continuous black line). Therefore, after the 8 seconds of 365 nm excitation, the photo-generation of the fluorescent excited state is complete. Gaussian bands decomposition of the emission spectrum reveals 4 bands with different intensity centered at about 480 nm, 504 nm, 525 nm, and 556 nm (dashed lines). Figure 1b shows the fluorescence spectrum of a single photoreceptor under 436 nm excitation light, after 10 seconds of excitation with the 365 nm light. A maximum fluorescence signal arises after the insertion of the blue-violet filter (continuous black line). Upon illumination by 436 nm light we can observe a fading of the green emission that decreases to almost zero after 10 seconds (continuous gray line). Regeneration of the non-fluorescent ground state is almost complete. Gaussian bands decomposition of the emission spectrum reveals 3 bands with different intensity centered at about 500 nm, 525 nm, and 556 nm (dashed lines). The bands obtained by the Gaussian decomposition indicate the presence of a mixture of very similar conformers in the photoreceptor, each one capable of photocycling between a non-fluorescent parent species and a fluorescent excited species. Figure 1c shows a very simple scheme of the photo-cycle of the Euglena photoreceptor: illumination at 365 nm of the non-fluorescent ground state leads to the photo-generation of the fluores-
406
V. Passarelli et al.
Fig. 1. a. Fluorescence spectrum of a photoreceptor excited at 365 nm; b. fluorescence spectrum of a photoreceptor excited at 436 nm; c. scheme of the photoreceptor photocycle. In a and b the dashed lines are Gaussian decomposition bands. See text for details.
Euglena photoreception
407
Fig. 2. a. Absorption spectra of the ground (continuous gray line) and excited (continuous dark line) state of the photoreceptor, showing the Gaussian decomposition bands (dotted lines), with superimposed absorption spectrum of the eyespot (dashed line); b. difference spectrum between the absorption spectra of the ground and excited state of the photoreceptor.
cent excited state, which in turn is driven back to the ground state by illumination at 436 nm (Evangelista et al. 2003). Figure 2a shows the absorption spectrum of the ground state (continuous gray line) of the photoreceptor (Gualtieri et al. 1989). Its structure is rather complex: at least two Gaussian bands can be detected in the α band, one centered at 456 nm, with a 15% area, and the other centered at 501 nm, with a 85% area (Fig. 2a, dotted lines). These two bands should correspond to the excited conformers (456 nm) and parent conformers (501 nm) of the
photoreceptive protein present inside the photoreceptor, already shown by the fluorescence spectra. Since we know the two wavelengths which induce
Fig. 3. Scheme of the photoreceptor photocycle showing the screening effect of the eyespot.
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V. Passarelli et al.
the transition from the ground state to the excited state (365 nm) and from the excited back to the ground state (436 nm), we can infer a difference spectrum between these two states. This difference spectrum shows a positive deviation corresponding to the wavelengths that cause the transition of the ground state to the excited state, around 365 nm, and a negative deviation corresponding to the wavelengths that cause the opposite transition, from the excited back to the ground state, around 436 nm. So far, we have not succeeded in measuring the absorption spectrum of the excited state of the photoreceptor, due to difficulties of setting-up instrumentation for measuring this kind of spectrum. However, a possible absorption spectrum of the excited state (Fig. 2a, continuous dark line) can
be obtained by inverting the areas of the two bands corresponding to the two conformer species of the photoreceptive protein, assigning the 85% area to the band centered at 456 nm, and 15% area to the band at 501 nm (Fig. 2a, dotted lines). In the excited state of the photoreceptor the excited protein conformer should be present in higher amount with respect to the parent conformer. Using the hypothetical absorption spectrum of the excited state of the photoreceptor, we obtain a difference spectrum that best fits the inferred difference spectrum: this spectrum possess a positive band centered around 365 nm and a bigger negative band centered around 436 nm (Fig. 2b). Figure 2a also shows the absorption spectrum of the eyespot (dashed line). The analysis of this spec-
Fig. 4. Example of the dynamics of the light alignment of Euglena obtained by mathematical simulation; e = eyespot; p = photoreceptor.
Euglena photoreception
trum shows that it can be superimposed on to the α band of the absorption spectrum of the excited form of the photoreceptor, with the same decomposition bands. We can use all the gathered spectroscopic information to interpret Euglena photo-responses. As the cell rotates while swimming (rotation frequency: 2 Hz; Ascoli et al. 1978), the photoreceptor experiences periodic shading by the eyespot, which comes between the light source and the organelle. This shading was believed to trigger the photophobic responses that regulate cell orientation. The classic interpretation of Euglena photobehaviour by Jennings (1906) considered a change in the intensity of light the determining cause of the cell reaction movements. A century later, our data allow us to improve and refine this model. It is not a change in light intensity that leads to the cell reorientation, but a selective screening of the absorption window of the excited state of the photoreceptor by the eyespot. Since the absorption spectra of the eyespot and that of the excited state of the photoreceptor are super-imposable, the screening effect of the eyespot leads to the interruption of the photo-cycle, preventing the transition of the excited state of the photoreceptor to its ground state (Fig. 3). During all the shading period, the photoreceptor is in the excited state, which probably generates a photoelectric signal due to the change in dipole orientations between the excited and parent conformers of the photoreceptive protein. Under the influence of this electric field, a displacement photocurrent could be propagated through paraxial rod filaments via charge transfer between rod proteins, which in turn generates longitudinal waves of contraction along the paraxial rod. The contractility of the rod should regulate the flagellum movement, turning the cell body toward the light source (Walne et al., 1998). The ground state of the photoreceptor is restored when the photoreceptor again achieves alignment with the light. An example of the dynamics of the light alignment of a model Euglena obtained by resolving two sets of equations, one relating to motion of the cell body and the other to motion of the flagellum (which space does not allow us to elaborate here), is shown in Figure 4. The initial orientation of the cell is opposite to the light direction, but in about 10 s, which correspond to 20 revolutions around
409
the cell main axis, Euglena is completely aligned with the light.
References Ascoli C., Barbi M., Frediani C. and Mure A. (1978): Measurements of Euglena motion parameters by laser light scattering. Biophys. J. 24, 585–599. Barsanti L., Passarelli V., Walne P. L., and Gualtieri P. (1997): In vivo photo-cycle of the Euglena gracilis photoreceptor. Biophys. J. 72, 545–553. Barsanti L., Passarelli V., Walne P. L., and Gualtieri P. (2000): The photoreceptor protein of Euglena gracilis. FEBS Letters 482, 247–251. Bogomolni R. A. and Spudich J. L. (1991): Archaebacterial rhodopsins: sensory and energy transducing membrane proteins. In: Spudich J. L., and Satir B. H. (eds.): Sensory Receptors and Signal Transduction, pp. 233–255, WileyLiss, New York. Cramer M. and Myers J. (1952): Growth and photosynthetic characteristics of Euglena gracilis. Arch. Mikrobiol. 17, 384–402. Evangelista V., Passarelli V., Barsanti L. and Gualtieri P. (2003): Fluorescence behaviour of Euglena photoreceptor. Photochem. Photobiol. 78, 93–97. Gabellieri E., Strambini G. B. and Gualtieri P. (1988): Tryptophan phosphorescence and the conformation of liver alcohol dehydrogenase in solution and in the crystalline state. Biophys. Chem. 30, 61–67. Gualtieri P., Barsanti L. and Passarelli V. (1989): Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis. Biochim. Biophys. Acta 993, 293–296. Gualtieri P., Barsanti L., Passarelli V., Verni F., and Rosati G. (1990): A look into the reservoir of Euglena: SEM investigation of the flagellar apparatus. Micron Microsc. Acta 21, 131–138. Jennings, H. S. (1906): Behavior of the lower organisms. Columbia University Press, New York. Knowles A. and Dartnall H. J. A. (1977): Photochemistry of extracted visual pigments. In: Davson H. (ed.): The eye Vol. 2B, The photobiology of vision pp. 289–345, Academic Press, New York. Marwan W. and Oesterhelt D. (1990): Quantitation of photochromism of sensory rhosopsin-I by computerized tracking of Halobacterium halobium cells. J. Mol. Biol. 215, 277–285. Pesce A. J., Rosen C. and Patsby T. L. (1971): Fluorescence spectroscopy, Marcel Dekker Inc., New York, USA. Stuart, J. A., D. L., Marcy, K. J., Wise and R. R. Birge (2003): Biomolecular electronic device applications of bacteriorhodopsin. In: Barsanti L., Evangelista V., Gualtieri P., Passarelli V. and Vestri S. (eds.): Molecular electronics: biosensors and biocomputers, pp. 265–299, Kluwer, Amsterdam. Verni F., Rosati G., Lenzi P., Barsanti L., Passarelli V., and Gualtieri, P. (1992): Morphological relationship between paraflagellar swelling and paraxial rod in Euglena gracilis. Micron Microsc. Acta 23, 37–44. Walne P. L., Passarelli V., Barsanti L. and Gualtieri, P. (1998): Rhodopsin: a photopigment for phototaxis in Euglena gracilis. Crit. Rev. Plant Sci. 17, 559–574.