Algal Eyes

Peter Hegemann, Institut fu r Biochemie, Regensburg, Germany Markus Fischer, Institut fu r Biochemie, Regensburg, Germany
Vision is defined in a very general sense as recognition of the ambient light pattern by a motile organism and its use for orientation in a local environment. The eye is the organ or organelle in which the light absorption and transformation into a transient intracellular signal occurs.

Secondary article
Article Contents
. Diversity of Algal Eyes . Function of Chlorophyceaen Eyes

Diversity of Algal Eyes

Motile microalgae are guided to places of optimal light conditions by means of their eyes, which constitute the simplest and most common visual system in nature. The eye contains the optics, the photoreceptor and elementary components of the signal transduction chain. Algal light antennae, which are used for detection of the light intensity, colour and direction of incidence, are more heterogeneous than animal eyes. They use not only dierent optical principles such as refraction, reection, absorbance and interference, but also dierent photoreceptors which may contain retinal, avin, pterins and phycoerythrin as chromophoric pigments. Since the chlorophycean eye is the best understood so far and shows the closest evolutionary relation to animal eyes, we will discuss this type in some detail and only touch upon other receptor designs. Algal eyes were classied on the basis of their functional concepts in a fundamental analysis by Foster and Smyth (1980).

Multilayer quarter wave stack antennas

The largest and most diverse order of green algae are the Chlorophyceae. Its most prominent and widely investigated member is the unicellular Chlamydomonas reinhardtii, which is schematized in Figure 1a. The alga is only 8 to 10 mm in diameter. The eyespot (which is also called stigma) is 1 mm in size and its orange pigmentation lies within the large chloroplast. Most chlorophyceaen algae have two active agella and they swim in a smooth helical pattern with the agella in a forward direction. During rotation the eye is scanning the environment for light intensity and quality. For receiving an interpretable light signal, the location of the eye is of fundamental importance. In most species the eye is found between agella and the equator of the cell. During rotation the eye advances the agellar beating plane by 20 to 408, the angle nicely matching the time needed for signal transduction from eye to agella. In the colonial alga Volvox carteri, the 2000 to 4000 somatics are embedded in the cell matrix at the surface of the spheroid. The somatic cells possess eyes and agella and are responsible for guiding the colony to places

that are optimal for photosynthetic growth. The 16 larger, eyeless and agella-less reproductive cells (gonidia) are located inside the sphere and have no contact with the extracellular medium. The cells in the front part of the colony contain bigger eyes with higher light sensitivity than the rear cells. Modulation of light incidence does not automatically provide, as implied above, a strong modulation of the perceived light signal, unless photon absorption by the photoreceptor molecules varies with the light incidence. In fully transparent cells with randomly distributed photoreceptors, the contrast is minimal and the light direction cannot be detected. In microalgae the photoreceptor is clearly localized (further discussed below) and an associated optical system provides the directivity. Due to the small eye size in most microalgae, a lens system would provide only refraction and scattering and would not be very benecial. Therefore, most microalgae developed optical systems which operate on the basis of light reection and constructive interference (Foster and Smyth, 1980). The chlorophyceaen eye consists of layers of hexagonally closed packed, carotenoid-rich lipid globules, which reect the light (Figure 1b). The reected light waves undergo constructive interference if the dierence of the light path of two reected light waves is a multiple of l/2 producing waves of interference maxima and minima. Reection and interference increase with the number of layers. Some algae, such as Chlamydomonas eugametos, Tetraselmis, Haematococcus or gametes of the seawater algae Acetabularia (order Dasyclades) and Ulva (order Ulvales), have only one or two layers. These eyes operate as quarter wave plates with relatively weak reection and front to back contrast. Eyes of Chlamydomonas reinhardtii or Hafniomonas reticulata may have up to eight layers. Consequently, the contrast between light incidence from dierent sites of the cell is much higher than in species with single-layered eyespots. Since the brightest interference maximum coincides with the eyespot overlaying part of the plasma membrane, the plasma membrane has been favoured as the ideal location of the photoreceptor (Foster and Smyth, 1980). Such a location provides a plausible means of communication with the agella, since the plasma
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Algal Eyes

Retinal switch Flagella Photoreceptor channel Ca2+ Mitochondrion t H+ Cation channel Green light Plasma membrane

Carotenoid vesicles

Vacuole Eyespot (stigma) Plasma membrane Nucleus

Chloroplast (a) (b)

125 nm (= /4)

125 nm (= /4)

Figure 1 (a) A scheme of a Chlamydomonas reinhardtii cell. (b) Magnification of the region squared in (a). The pigmented eyespot functions as an optical device (quarter wave stack), which together with the rhodopsin, the transducer (t) and the ion-conducting protein components form the functional eye. Note that in this diagram the rhodopsin and the photoreceptor channel form a single multimeric complex.

membrane is continuous with the agellar membrane. The half beam width of the antenna decreases with the number of carotenoid layers from 1308 in a single layered eye towards 608 in a multilayered system. This spatial resolution is further improved by the concave curvature of the eye and the orientation of the receptor molecules within the eyespot membrane. But nevertheless, algal eyes are designed for tracking diuse light instead of point light sources. Since reection and especially interference depends on the colour of the light, algal eyes are optimized for a certain colour range which is smaller than the range of a lens system. For wavelengths shorter than the optimum, interference occurs at a tilted angle whereas for longer wavelengths it is just reduced. In other words optimal light incidence is rare as in any other visual system. The light signal is colour-modulated during cell rotation and it shifts to blue when the cell is approaching the light source.

protein, which gives them a high refractive index. This makes them act as a wave guide, especially for light that strikes them under at angles. How the information received is transferred to the agella is completely obscure and hard to imagine.

Euglenophyceae: absorbing screens and dichroic crystal detectors

The second widely investigated alga besides C. reinhardtii is Euglena gracilis (Figure 2). Phototaxis in Euglenophyceae appears to have originated independently of that of the Chlorophyceae. The photoreceptor is the paraagellar swelling (PFS) of the long and active agellum at its base. It is physically shielded by the reservoir in which the agella root. The most fascinating property of this receptor is its crystalline nature (Piccinni and Mammi, 1978; Figure 2). The molecules of the crystal are oriented in a xed angle with respect to the antenna screen, which has strong implications for the directivity of the antenna for polarized and unpolarized light as well as for communication with the agellum. Optical diraction of transverse sections presents an orthogonal crystal lattice. The screen at the back of the antenna is the stigma, a pigmented mass of 3 to 7 mm within the cytoplasm on one side of the PFS. The stigma is not dichroic since its granules show spheric birefringence, characteristic of a radial array of the molecules. The net directivity is caused by a combination of an oriented, and therefore dichroic, receptor and an eective absorbing screen.

Cryptophyceae: dielectric slab wave guide antennas

Antennae of Cryptophyceae are located in the centre of the cell as a specialized part of the chloroplast. At its inner longitudinal surface there are about 35 pigment granules which absorb most strongly when the light is perpendicular to the swimming axis of the cell. Phycoerythrin, the pigment implicated by the action spectrum as the receptor for phototaxis, presumably lies within the thylakoid discs perpendicular to both the plane of the pigment granules and the long axis of the cell. The discs contain lipid and
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Algal Eyes

Paraflagellar swelling (PFS) with photoreceptor Photoreceptor crystal Short flagellum Reservoir

Eyespot (stigma) Nucleus Chloroplasts Long flagellum (with axoneme)

Long flagellum (a) (b)

Eyespot (stigma)

Axoneme

Photoreceptor crystal

(c)
Figure 2 (a) Schematic representation of Euglena gracilis. (b) Magnification of the region squared in (a). Longitudinal section through the eye. The unstructured eyespot granules outside the chloroplast serve as a shading device. The paraflagellar swelling (PFS) houses the photoreceptor system which, most likely, uses pterins for light harvesting and flavins as the functional redox component. (c) Cross-section through the flagellum with attached photoreceptor. The photoreceptor molecules are highly ordered, forming a monoclinic photoreceptor crystal.

Absorbing screens and noncrystalline flagellar receptors

Although antennae of Chrysophyceae, Xanthophyceae and Phaeophyceae have a design related to euglenophyceaen antennae, they dier in several fundamental respects: the eyespot droplets are formed within rather than outside the chloroplast; the paraagellar swelling is associated with the short, nonbeating secondary agellum; and the swelling is not crystalloid.

Rhodopsins versus flavins as functional photoreceptors

Since phototaxis, i.e. the orientation towards a permanent light source, includes directivity, the optical properties of

the eye and other shading pigments contribute to phototaxis action spectra to a larger extent than to those for phobic responses (behavioural responses to sudden changes in light intensity). Since phototaxis is measured in continuous light, several parameters such as adaptation phenomena, photochromic properties of the photoreceptors, and screening pigments distort the action spectra, especially when measurements are carried out at high light intensities. Consequently, phototaxis action spectra constructed from recordings at low irradiance reveal the nature of the behavioural photoreceptor in these algae in a much better way than high intensity spectra. Threshold phototaxis action spectra for Chlorophyceae are rhodopsin-shaped with maxima between 460 and 560 nm (Foster et al., 1984). These phototaxis spectra closely match the action spectra for ash-induced nondirectional photophobic responses.
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Algal Eyes

The rhodopsin idea should not be over generalized. More than 20 action spectra have been published for the phototaxis of euglenophyceaen algae. However, low intensity action spectra have been carefully established only by Diehn (1969). His action spectra for phototaxis and phobic responses in Euglena are avin-like spectra. Thus, the dominant photoreceptor for photon capture at low light levels seems to be a avin or a pterin/avin tandem. The uorescence of the PFS has been extensively studied in vivo and in PFS preparations. The major ultraviolet (UV) photoreceptor, P355, only weakly uoresces in the green. But, blue light excitation of P355 creates a bright uorescent species, P470, which exhibits an emission maximum near 520 nm (Barsanti et al, 1997; Ha der and Lebert, 1998). These data lead to the following conclusions. The PFS contains a pterin as the dominant light harvesting chromophore. The pterin transfers the energy to a second, avin type chromophore (P470), which converts the light energy into a agellar signal. The dual receptor system may operate similarly to higher plant blue light receptors (cryptochromes). Unfortunately, so far no molecular information is available about any of the photoreceptor components.

Function of Chlorophyceaen Eyes

The rhodopsin chromophore
The claim of a rhodopsin type photoreceptor in chlorophyceaen algae was substantiated by using blind retinaldecient Chlamydomonas cells. After complementing the decit by addition of retinoids or various retinal analogues, phototaxis and photophobic responses were tested. The major conclusions are summarized by the following: whereas in animal rhodopsins 11-cis retinal or the derivatives 11-cis-3-hydroxy, 11-cis-4-hydroxy, or 11-cis 3,4-dehydroretinal form the functional chromophore, chlamyrhodopsin contains an all-trans,6-S-trans retinal chromophore very much like all archaeal rhodopsins. During illumination the chromophore undergoes a 13trans to cis isomerization. The isomerization is converted into a conformational change of the protein via the 13methyl group. The retinal is in a planar conformation across the C6C7 single bond, which links the trans conformation of the polyene chain to its ionone ring. The ring accelerates reconstitution, but a chromophore with only three conjugated double bonds plus methyl groups can provide photosensitivity (Spudich et al., 1995).

Ion channel activation

Light excitation of the rhodopsin photoreceptor in chlorophyceaen algae results in the generation of a cascade of electrical events. The analysis of rhodopsin-induced
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photocurrents became possible by application of a suction pipette technique, rst, in a pioneering study, to Haematococcus pluvialis (Litvin et al., 1978) and many years later to cell-wall-decient Chlamydomonas reinhardtii cells (Harz and Hegemann, 1991). Only recently, photocurrents were recorded from individual cells of a Volvox carteri dissolver mutant (Braun and Hegemann, 1999). The photocurrents are a composition of several individual components which may be attributed to at least three conductances. A Ca2 1 conductance, CCa, in the eyespot region is activated with virtually no delay ( 5 50 ms). It is graded with the ash energy in all three studied algal species. In C. reinhardtii it peaks within 1 ms, and decays within 20 ms in a voltage-dependent manner. In V. carteri, the rise and the decay are four to six times slower, accounting for the much slower rotation frequency of the intact spheroid, compared to its unicellular counterparts. CCa saturates only at high ash intensities in parallel with the rhodopsin bleaching. CCa is always transient and, in continuous light, it decays to a level of only a few per cent of the maximum. In addition, the rhodopsin activates a proton conductance, CH, which dominates the photocurrent at low light levels. Since at low light, the delay between ash and the beginning of the photocurrent rise is long, and the amplitudes are large, an amplication system is required for the activation of CH. The current model is explained in Figure 1b. The rhodopsin is part of a low conductance ion channel complex, CCa, which is activated upon light absorption and retinal isomerization. The Ca2 1 inux depolarizes the cell and the depolarization is sensed by agellar ion channels. In addition, the rhodopsin activates a proton conducting ion channel, CH, via a transducer molecule, t. More than one proton channel is activated per rhodopsin. CH saturates at relatively low light levels. The low intensity response is likely to be propagated by intracellular transduction processes from the eye to the agella. A K 1 eux repolarizes the cell after a light ash towards the K 1 equilibrium potential. In continuous light, the electrical responses are more complex and poorly understood. Stationary photoreceptor currents appear. But they are accompanied by agellar current and are dicult to study separately. These steady state currents deserve more attention because modulated stationary currents constitute the basis for phototaxis at high light levels. When in unicellular species the integral of the photoreceptor current exceeds a critical level, agellar currents are triggered. In C. reinhardtii and H. pluvialis a fast action potential-like FF current is observed. FF is the trigger for the photophobic response, during which the cells swim backwards for some hundred milliseconds, in order to escape suddenly appearing regions of harmful bright light. Since unicellular agellates are nearly isopotential, the agellar conductance, CF, senses the primary depolarization, caused by the two eye conductances CCa and CH.

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Algal Eyes

Thus, no signal amplication and no transmitter are required for its activation. However, at low intensity ashes when no FF currents appear, modulation of the agellar beat frequency and beating plane must be caused by intracellular signalling all the way from the eye to the agellar base.

The opsin primary structure

As the rst member of the algal opsin family, chlamyopsin was biochemically identied by labelling the protein in a C. reinhardtii retinal-decient mutant using 3H-retinal. Later, eyespot membranes were prepared from wild type cells, and the rhodopsin absorption was identied in these fractions using dierential microspectrophotometry (Beckmann and Hegemann, 1991). However, the excess of carotenoids was so high that the chromophore could not be characterized in any detail. But nevertheless, the opsin was puried to homogeneity from this source (Deininger et al., 1995). The opsin is sharply conned to the eyespot area. Early in the division cycle the eyespot pigmentation disappears, but the opsin spot of the mother cell persists until division is fully completed, and is still visible at the time when the two new eyes are formed in the daughter cells. The only algal opsin sequences available so far are those from C. reinhardtii and V. carteri. Their gene structure is similar. The coding regions are interrupted by seven introns. Six introns in both genes are at the same position. Only intron 7 (I7) is located further downstream in V. carteri. Size and sequences of the introns vary to a much larger extent than the coding region. It is remarkable that the algal introns I2 and I4 are positionally conserved in opsin genes from vertebrates, arthropods and cephalopods. At the position of the algal I7, an insertion is found in all animal opsin genes. It follows that the algal I2, I4 and I7 predate the plant animal divergence. The algal opsin genes code for proteins with molecular weights of around 26 kDa. They are the smallest eukaryotic opsins so far known. Volvoxopsin and chlamyopsin are 65% identical and certainly belong to the same protein family. The algal opsins are unique in many respects. First of all, the proteins are highly charged. The seven transmembrane segments typical for the whole opsin superfamily cannot be identied in the algal opsins. Thus, their topography is still unclear. No signicant homology was found between algal and archaeal opsins, although their chromophoric properties are so similar. However, algal opsins show sequence homology to animal opsins with a slightly closer relation to invertebrate than to vertebrate opsins. Compared to animal opsins, algal opsins are truncated at both ends. But more than half of the amino acids, which are identical in all or most invertebrate opsins, are also present in the algal opsins. Chlamyopsin is most related to Calliphora opsin

(23% identity), whereas volvoxopsin is mostly related to Drosophila rhodopsin 6 (DROME rh6, 21%). Solely from the sequence there is no clear indication for an interaction of the opsins with G proteins. The tripeptide Asp-Arg-Tyr or Glu-Arg-Tyr at the cytoplasmic border of the third transmembrane segment in G-protein-coupled receptors and a characteristic feature for G protein interaction is missing in the algal proteins. On the other hand, the sequence comprising the third cytoplasmic loop between the transmembrane segments 5 and 6 in invertebrate opsins, which also plays a dominant role for G protein recognition, shows strong homology to the corresponding algal segments. G proteins have been identied in eyespot preparations of C. reinhardtii and Spermatozopsis similis. In S. similis the GTPase activity was shown to be green light-dependent and possibly rhodopsin-controlled (Calenberg et al., 1998). From these biochemical experiments it is not unlikely that algal opsins, despite their unusual sequences, do activate G proteins. The comparison of the opsin gene structure and the derived amino acid sequences may be reconciled to the hypothesis that algal opsin genes reect the ancient exons, from which all known eukaryotic opsin sequences, including pinopsin, melanoopsin and retinochrome, had developed in a sequential and modular process. In contrast to animal opsins, algal opsins exhibit characteristics that suggest their direct participation in a receptorion channel complex. The lysine-rich sequences interspaced by hydrophobic amino acids (5880) are reminiscent of S4 stretches of voltage-gated or cGMPgated channels. In addition, the sequence motif Val-SerLeu-Lys-Ser-Thr-Val-Gly-Ile187195, located between the potential transmembrane segments, is reminiscent of the pore loop region of voltage-gated potassium channels. In summary, if the rhodopsins form ion channel complexes and, in addition, are coupled to G proteins, this would elegantly explain the activation of two dierent conductances within the eyespot area.

References
Beckmann M and Hegemann P (1991) In vitro identication of rhodopsin in the green alga Chlamydomonas. Biochemistry 30: 3692 3697. Barsanti L, Passarelli V, Walne PL and Gualtieri P (1997) In vivo photocycle of the Euglena gracilis photoreceptor. Biophysical Journal 72: 545553. Braun F-J and Hegemann P (1999) Two independent photoreceptor currents in the spheroidal alga Volvox carteri. Biophysical Journal 76: 16681678. Calenberg M, Brohsonn U, Zedlacher M and Kreimer G (1998) Lightand Ca2 1 -modulated heterotrimeric GTPases in the eyespot apparatus of a agellate green alga. Plant Cell 10: 91103. Deininger W, Kro ger P, Hegemann U, Lottspeich F and Hegemann P (1995) Chlamyrhodopsin represents a new type of sensory photoreceptor. EMBO Journal 14: 58495858. Diehn B (1969) Action spectra of the phototactic responses in Euglena. Biochimica Biophysica Acta 177: 136143.

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Foster KW and Smyth RD (1980) Light antennas in phototactic algae. Microbiological Reviews 44: 572630. Foster KW, Saranak J, Patel N et al. (1984) A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eucaryote Chlamydomonas. Nature 311: 756759. Ha der D-P and Lebert M (1998) The photoreceptor for phototaxis in the photosynthetic agellate Euglena gracilis. Photochemistry and Photobiology 68: 260265. Harz H and Hegemann P (1991) Rhodopsin-regulated calcium currents in Chlamydomonas. Nature 351: 489491. Litvin FF, Sineshchekov OA and Sineshchekov VA (1978) Photoreceptor electric potential in the phototaxis of the alga Haematococcus pluvialis. Nature 271: 476478. Piccinni E and Mammi M (1978) Motor apparatus of Euglena gracilis: ultrastructure of the basal portion of the agellum and the paraagellar body. Bollettino della Zoologica 45: 405414.

Spudich JL, Zacks DN and Bogomolni RA (1995) Microbial sensory rhodopsins: Photochemistry and function. Israel Journal of Chemistry 35: 495513.

Further Reading
Hegemann P (1997) Vision in microalgae. Planta 203: 265274. Kreimer G (1994) Cell biology of phototaxis in agellate algae. International Review of Cytology 148: 229311. Land MF (1972) The physics and biology of animal reectors. Progress in Biophysical and Molecular Biology 24: 75106. Sineshchekov OA and Govorunova E (1998) Rhodopsin-mediated photosensing in green agellate algae. Trends in Plant Science 4: 58 63. Witman GB (1994) Chlamydomonas phototaxis. Trends in Cell Biology 3: 403408.

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