668

J. Phycol.

37,

668–676 (2001)

MINIREVIEW

ALGAL SENSORY PHOTORECEPTORS

1

Peter Hegemann,

2

Markus Fuhrmann, and Suneel Kateriya

Institut für Biochemie I, Universität Regensburg, 93040 Regensburg, Germany

Sunlight is the primary energy source for all life onearth, but it also plays an important regulatory rolefor the growth and development of living organisms.Additionally, light is used as a source of information,which enables the organism to orient and adapt to itssteadily changing world. To exploit light as a complexsensory stimulus, algae and higher plants developedsophisticated networks of photoreceptors and sensorypathways that generate appropriate responses. Theseresponses cover a time scale of seconds up to days oreven years.

the principal photoreceptors in nature

Nature uses a very limited number of principal or-ganic molecules to form sensory photoreceptors thatcontrol developmental and visual processes (Fig. 1).Different photoreceptors evolved in different evolu-tionary branches because of the needs of organismswith respect to available light quality, light quantity,and the necessary response time and amplificationrange of the sensory signal.

Rhodopsins.

The classic and most carefully studiedsensory system is vision in higher animals. The photo-

receptor is rhodopsin with retinal (Fig. 1a) as the chro-mophoric group. In all rhodopsins known so far, reti-nal is bound to a lysine residue of the apoprotein(opsin) via a Schiff-base. Dark-adapted animal rhodop-

sins (type II rhodopsins) contain a twisted 11-

cis

retinalthat isomerizes after light excitation into all-

trans

, thustriggering a conformational change that initiates the

signaling process. Retinal absorption can be fine-tunedby amino acid charges of the retinal pocket, which al-

lows the entire spectrum between 360 and 635 nm to becovered (Kleinschmidt and Harosi 1992, Kochendoerferet al. 1999). Due to the high transmission of only blue-green light in water, however, most rhodopsins absorbaround 500 nm (Goldsmith 1991). Rhodopsins are

also used in the archaeal branch (type I rhodopsins),where they serve as sensory photoreceptors for orienta-tion of the cells in different light qualities (sensoryrhodopsins) or as light-driven ion transporters (bacterio-rhodopsin and halorhodopsin). Archaeal rhodopsins

contain all-

trans

,15-S-anti retinal, which upon light ab-

sorption undergoes a concerted 13-

cis

isomerization.The concomitant rotation of the N-H dipole inducesion movement across the retinal barrier, resulting in a

pumping process in bacteriorhodopsin and halorhodop-sin (Kolbe et al. 2000). The function of the sensoryrhodopsins is surprisingly similar. A proton within theretinal binding site is displaced after retinal isomeriza-tion; however, the proton is not released but insteaddrives conformational changes within the rhodopsinand the attached transducer protein (Htr) (Spudich1994). Recently, an archaeal type I rhodopsin sequencehas been discovered in the fungus

Neurospora

(Bieszkeet al. 1999), but its function is not yet known.

In general, rhodopsins are employed by motile or-ganisms that must respond rapidly on a time scale ofmilliseconds to seconds as environmental conditionschange or as the organism changes position relativeto its static surroundings. Rhodopsin-based systemsare fast, and the intracellular response is immediatelyreset so that the system is prepared for a new light in-put. Thus, rhodopsins should be expected to occur inmotile microalgae (or gamete and zoospores) and notin sessile macroforms.

Phytochrome.

The classic sensory photoreceptor ofhigher plants is phytochrome, which contains phytochro-mobilin as the chromophoric group (Fig. 1b). The lineartetrapyrrole undergoes a sequential double isomeriza-

tion (C15-Z,14

syn

to C15-E,14

anti

), which includes a rota-tion of ring D (Andel et al. 1996). The two photoconvert-ible isoforms (

Z

and

E

) are both stable in the dark. Thus,phytochromes alternate between two molecular stagescreating a long-lasting signal within an individual cell.Two stable stages of a single photoreceptor species ap-pear insufficient to control the complex development ofa higher plant under a broad range of heterogeneous en-vironmental conditions, however, and higher plants con-tain up to 5 phytochromes (A–E), each with slightly dif-ferent signaling properties (Sharrock and Quail 1989).Phytochromes are light-regulated serine/threonine pro-tein kinases (Yeh and Lagarias 1998), but their signaltransduction pathway has not been elucidated in detail.Phytochromes are ancient molecules that might have de-veloped from a more compact light sensor in cyanobacte-ria (blue-green algae) (Hughes et al. 1997). In contrast

1

Received 14 May 2001. Accepted 6 August 2001.

2

Author for correspondence: e-mail peter.hegemann@biologie.uni-regensburg.de.

Key index words:

cryptochrome; gamete release; ga-metogenesis; opsin; phototaxis; phototropin; phyto-chrome; rhodopsin; rhythmicity

Abbreviations:

Cop, chlamyopsin; Cry, cryptochrome;FMN, flavin mononucleotide; Phot, phototropins;TM, transmembrane

ALGAL SENSORY PHOTORECEPTORS

669

to the rhodopsins, the phytochrome superfamily is quitehomogeneous and the primary steps of all phyto-chrome-signaling systems seem to be similar. Phyto-chromes might contribute to developmental processes inseveral algal species but preferentially in those that liveon shores or in shallow depth because of the efficient ex-tinction of red light in sea water (Goldsmith 1991).

Photoactive yellow protein.

Negative photophobic re-sponses and photoaccumulation of the halophilic pur-ple bacterium

Ectothiorhodospira halophila

are thought tobe caused by the photoactive yellow protein (PYP), socalled for its brilliant yellow color. PYP from

E. halophila

is a soluble protein of only 14 kDa with a primary struc-ture related to the widely distributed PAS domains. PYPcontains a 4-hydroxycinnamate chromophore (Fig. 1c)that is covalently attached to a thiol group located inthe hydrophobic core of the protein (Baca and Get-zoff, 1994). The chromophore undergoes C7,8

cis-trans

isomerization in concert with a flipping aroundthe C-S single bond of the thioester. Subsequently thephenylate anion picks up a proton from its protein en-vironment thus inducing the conformational change ofthe protein (for review see: Schlichting and Berendzen1997, Heberle and Gensch 2001).

Cryptochrome and phototropin.

Responses to blue lightin living organisms belong to the earliest characterizedbut still poorly understood phenomena in photobiology(Darwin 1881). The changes induced in higher plantsby blue light are not identical to those mediated by redlight. The responsible cryptic chromophores remainedunidentified until 1993 when M. Ahmad and A. Cash-more discovered the molecular defect of the hy4

Ara-bidopsis

mutant (Ahmad and Cashmore 1993). Theyshowed that the responsible cryptochrome (Cry) is re-

lated to class I photolyases, which are enzymes that re-pair cyclobutane pyrimidine dimers by using UV lightas an energy source. Cry1 photoreceptors participatein uncounted developmental processes in coopera-tion with the red-light receptor phytochrome. Later, asecond cryptochrome (Cry2) was identified that con-trols flowering time and is responsible for measuringday length (Guo et al. 1998). The flavin adenindinu-cleotide (FAD, Fig.1d) chromophore of Cry1 andCry2 is photoreduced in a primary activation step. Be-cause the reduced active species, FADH, exhibits asmall extinction coefficient, Cry needs a second chro-mophore for efficient light harvesting, which is eithermethylene-tetrahydrofolate (MTHF) (Fig.1e) or dea-zaflavin (8-HDF). Cryptochromes normally exhibitfine-structured spectra with maxima near 370 nm and450 nm, but, in some cases, the semichinone radicalcauses an additional absorption at 550 nm, often mis-interpreted as an independent green light receptor.Cryptochromes operate as light-regulated protein ki-nases. Meanwhile

cry

genes were also identified in hu-man beings and mice (Devlin and Kay 1999). In bothsystems Cry-proteins directly interact with the circa-dian clock but whether these Crys function as sensorylight receptors is questionable (Foster 2001a). Crypto-chrome-related genes and proteins have also beenidentified in

Neurospora

(White collar one [WC1]).WC1 is part of the clock, and the evidence is quiteconvincing that WC1 is also a photoreceptor for thelight input pathway (Crosthwaite et al. 1997). Basedon current information cry-related flavin photorecep-tors are the most widely distributed light sensors innature, but the detailed photochemical reaction andthe down-stream signaling processes are not known.

Fig. 1. Principal chromophoric compoundsused in sensory photoreceptors. The photorecep-tors carrying the respective chromophores are in-dicated in brackets. The arrow in b indicates thesite where phytochromobilin reacts with the pro-tein. MTHF � N5, N10-methenyl-tetrahydrofolate(a pterine derivative ).

670

PETER HEGEMANN ET AL.

Phototropins (now named Phot), the photorecep-tors for phototropism in higher plants and fungi, arethe second class of flavin-based photoreceptors. Photuse two flavin mononucleotides (fully oxydized FMN,Fig. 1d) as chromophores and constitute a classic serine/threonine kinase domain (Briggs and Huala 1999, Briggset al. 2001). Recently, evidence appeared that Phot arealso responsible for chloroplast relocation (Jarillo et al.2001, Kagava et al. 2001).

algal photoreceptors: chloroplast movement

In the second half of the 19th century, botanistsdiscovered that displacement and reorientation of thechloroplast in the cell is regulated by light (Böhm1856). The action spectrum as well as the reversiblered/far-red antagonism favored phytochrome as thephotoreceptor pigment. The peculiar morphology ofthe alga

Mougeotia scalaris

allowed a partial irradiationof the cell with microbeams, and this work resulted inthe conclusion that the photoreceptor is located inthe cytoplasm. In

Mougeotia

one phytochrome genehas been found so far (Winands and Wagner 1996).Expression of this gene

in vivo

is light-regulated, theexpression being slowed down by P

fr

, the active formof phytochrome. Phytochrome, in addition to moni-toring the basic light quality and intensity, detects thedirection of the impinging light, which may or maynot be linearly polarized. An algal phytochrome pro-tein was isolated from the green alga

Mesotaenium cal-doriorom

, in which a function as a sensor for chloro-plast movement was also implied (Kidd and Lagarias,1990). Around 2

�

10

5

molecules of the 120 kDa pro-tein are present in the algal cytoplasm. The absorptionmaxima of 650 nm and 722 nm for P

r

and P

fr

are blueshifted compared to those from angiosperm tissues.

Besides the typical main phytochrome peak in redlight, the action spectrum for chloroplast movementin

Mougeotia

has a smaller peak in the blue region(Haupt 1959). Moreover, against a background ofstrong far-red light the response can no longer be in-duced by red light, whereas the blue-light effect is notimpaired. The flavin action spectrum recorded in thepresence of red background light is strong evidencefor a separate cryptochrome- or phototropin-type pho-toreceptor. A clear example appears to be the diatom

Pleurosira laevis

, in which chloroplast movement is effi-ciently mediated mainly by blue and green light (Fu-rukawa et al. 1998). In summary, it may be concludedthat chloroplast movement in many algal species ismediated by a phytochrome/cryptochrome or a phy-tochrome/phototropin pair of photoreceptors. How-ever, photoreceptor mutants, a gene disruption sys-tem, or antisense transformants are needed to supportthis hypothesis.

Chloroplast development.

In most algae, proteins of thephotosynthetic apparatus (or their respective RNAs)are down-regulated if the cells are kept in darkness forlonger times and up-regulated after they are broughtback into the light. This is because the photosyntheticunits are simply not needed during longer dark peri-

ods and have to be highly expressed under low-to-mod-erate light conditions. In several algae, blue light is amore efficient upregulator of the photosynthetic appa-ratus than red light, and this regulation is independentof photosynthetic activity. The only system studied on amolecular level is again

Chlamydomonas reinhardtii

, butthe data are not completely consistent. Blue light en-hances the transcription of several genes encoding en-zymes of chlorophyll and heme biosynthesis (Mattersand Beale 1994, 1995a, 1995b). Blue light also regulatesthe ratio of the light-harvesting complex LHCII to itsreaction center RCII by stimulating the transcriptionof the some of the 7 genes encoding the chl

a

/

b

bind-ing proteins (

lhcb

, formerly named

cab

) (Shepherd et al.1983, Johanningmeier and Howell 1984). In additionlight controls expression of many enzymes of the pen-tose phosphate pathway (Calvin cycle).

The Lhcb homologues in heterokont chromophyticalgae including diatoms, brown algae and chrysophytesare the fucoxanthin-chl-

a/c

-binding proteins (FCPs).The

fcp

genes are light-inducible in dark-adapted algaeand expression responds to a range of different lightqualities. In

Thalassiosira weissflogii

, cryptochrome-, rhodop-sin-, and phytochrome-based sensing systems may allbe present (Leblanc et al. 1999). An inhibitory role ofblue light for chloroplast development at high-lightlevels has been most clearly shown for the brown mac-roalga

Macrocystis pyrifera

, but details are not availableyet. However, it is clear that the inhibition is not aphotosynthetic effect, because red light is ineffective(Apt et al. 1995). Clear, low-intensity action spectrafor any aspect of chloroplast development in algae areabsent, and information about the nature of the blue-light receptor is not available; however, the light in-duction of

gsa

in

C. reinhardtii

is inhibited by the flavinantagonist diphenyleneiodonium. Moreover, in reti-nal-deficient cells, blue-light regulation of

gsa

is stillpossible, which excludes the contribution of rhodop-sin and favors the participation of a flavin-based crypto-chrome (Herman and Im 1999). In summary, there islittle doubt that expression of genes encoding light-harvesting complexes is caused by one or more blue-light receptors, whereas it is premature to concludethat phytochrome and even rhodopsin are involved.One gene is now available that codes for a blue-lightreceptor candidate that might mediate the regulatoryrole for chloroplast development. It is the crypto-chrome homologue Cph1 identified by Small et al.(1995). The hypothetical receptor is up to 49% identi-cal to Cry1 of

Arabidopsis thaliana

and contains the typ-ical binding sites for FAD and 5-MTHF, the structuresof which are seen in Figure 1. In

C.reinhardtii

the en-coded protein is only present in darkness but rapidlydegrades in the light. However, overexpression didnot result in a clear phenotype (N. A. Reisdorph andG. D. Small, University of South Dakota, personal com-munication) and knock-out mutants are required tounderstand the function.

One has to be careful before all light-induced devel-opmental processes are assigned to special sensory

ALGAL SENSORY PHOTORECEPTORS

671

photoreceptors. The synthesis of certain chloroplastproteins involved in photosynthesis—especially of thereaction center protein D1—is activated 50- to 100-foldin response to light without an increase of the corre-sponding mRNA levels (Fromm et al. 1985). The sce-nario is governed by a redox-dependent disulfideisomerase that modulates binding affinity of the chlo-roplast polyadenylate binding protein (cPABP) to the5

�

-UTR of the psbA mRNA (Kim and Mayfield 1997).At high light intensities photosynthesis is down-regu-lated to prevent photooxidation. Shifting

Dunaliellatertiolata

from high- to low-light conditions leads to arapid induction of genes encoding the chl

a

/

b

proteinsof the light harvesting complex II (

lhcb

, LaRoche et al.1991). It has been proposed that this increased produc-tion of light harvesting pigment complexes under low-light conditions is controlled by redox signaling fromthe chloroplast to the nucleus (Escoubas et al. 1995).

developmental processes

Under a standard light-dark cycle, a culture of thespheroidal alga

Volvox carteri

develops in a preciselysynchronized way. Embryogenesis occurs during thedark period, but somatic cells and gonidia remain im-mature. The final differentiation only occurs after thelight comes back on. Light triggers virtually instanta-neous translation of various preexisting mRNAs. Theaction spectrum for the resulting protein synthesis isclearly rhodopsin-shaped with the maximum in thegreen region of the spectrum (530 nm). This is thefirst developmental process for which rhodopsin wasclearly indicated as the responsible photoreceptor(Kirk and Kirk 1985). The development of many mac-roalgae is controlled by light, and I would expect thatthis is true for many of them. Blue light is most effec-tive in most cases, but there is no single case in whichthe nature of the photoreceptor can be clearly de-rived from a clear action spectrum and little progresshas been made in this respect recently. Therefore,there is not much to add to the review of Dring (1988).

Gametogenesis and gamete release.

Gamete formation inunicellular algae is often induced when the nitrogenavailability becomes limiting to growth. As early as 1956,Lewin observed that the formation of mating compe-tent

C. reinhardtii

gametes depends on light. Many yearslater, Treier et al. (1989) demonstrated that this com-petence is mediated by a sensory photoreceptor thatabsorbs mainly in the blue region of the spectrum andnot by photosynthesis. The lack of nitrogen serves as atrigger to initiate the program of gametic differentia-tion and only after so called pregametes have beenformed, 90 min of blue light irradiation provides fullmating competence (reviewed by Beck and Haring1996). The maxima of the action spectrum for game-togenesis at 370 nm and at 450 nm are highly indica-tive for a flavin-type sensory receptor (Weissig andBeck 1991). The fact that long irradiation is necessarybut the

C. reinhardtii

Cry-homologue Cph1 is rapidlydegraded in the light (Reisdorph and Small, personal

communication) makes it unlikely that Cph1 is the re-sponsible regulator for this process. The only candi-date in sight at the moment is a phototropin homo-logue, Phot (Briggs et al. 2001), which recently appearedas a partial cDNA sequence, expressed sequence tag(EST), from a sequencing project (Asamizu et al. 1999).The encoded protein contains two FMN-binding do-mains very related to the phototropin from

A. thaliana

and other higher plants, and the overall structure is alsosimilar (64%) (K. Huang and C. F. Beck, University ofFreiburg, Germany, personal communication). Numer-ous blue-light responses have been described in brownalgae (reviewed by Dring 1987). One clear example isgametogenesis in

M. pyrifera

(Lüning and Neushul 1978)suggesting that light is also needed for gametogenesis ofmacroalgae.

Gamete-release from gametangia of the marine alga

Bryopsis plumosa

represents another blue-light regulatedresponse. Mine et al. (1996) determined an action spec-trum for this discharge resembling two major peaks at370 and 450 nm, also pointing to a flavin-based, blue-light receptor. In contrast, in

Ulva mutabilis

, the gameterelease is regulated by a swarming inhibitor, which isproduced during gametogenesis (Stratmann et al. 1996)and by red light (Wichard 2001).

phototropism

Phototropism has been studied in several macroal-gae, but action spectroscopy has been carried out inonly a few species. The clearest results came from re-generated segments that were cut from the tip of athallus. Bending towards the light occurs below thetip of the regenerating thallus. The qualitative highand equal intensity spectrum peaks at 467 nm (Isekiet al. 1995a). The new rhizoids bend away from lightunder almost all light conditions (Iseki et al. 1995a).The equal response spectrum should be quantitativelymore precise with maxima at 370, 410 and 465 nm(Iseki et al. 1995b). Both spectra suggest a photore-ceptor with flavin as the only chromophore, and aphototropin-like Phot1 receptor would be most ap-propriate for both phototropic processes. Taken to-gether, the findings of blue-light dependent gameto-genesis and of positive and negative phototropism ofdifferent cell types of

Bryopsis

point to the existence ofan elaborate system of photoreceptor signaling path-ways in marine macroalgae. In macroalgae the releaseof spores is commonly stimulated by blue light (re-viewed by Dring 1988), whereas in

Laminaria

bluelight delays the release (Lüning 1981).

Photomovement responses.

Phototactic responses havebeen observed in hundreds of algal species includinggreen algae, dinoflagellates, and brown algae (Foster andSmyth 1980). Phototaxis depends on the ability of an or-ganism to detect the direction of the incident light, whichrequires a directional light antenna. Many unicellular al-gae such as

C. reinhardtii

,

Haematococcus pluvialis

, or

Du-naliella salina

possess pigmented eyespots, which functionas directional antenna that together with the photorecep-

672

PETER HEGEMANN ET AL.

tor and the downstream signaling system forms the func-tional eye. These eyes are among the smallest directionalantennas found in nature (Melkonian and Robenek1984). The antenna is based on layers of carotenoid lipidglobules, which provide the directivity by reflection andconstructive interference (Land 1972, Foster and Smyth1980, Hegemann and Harz 1998). Algal eyes are only

1

�

m in diameter. During rotation (1–2 Hz) the eye ad-

vances the flagellar beating plane by 20 to 40

�

degrees,providing a time window of roughly 100 ms for the signalto propagate from the eye to the flagella.

rhodopsins as photoreceptor candidates

Many of the phototaxis action spectra recorded fromunicellular algae or gametes are rhodopsin shaped if cor-rectly analyzed. Among these are the chlorophycean al-

gae

Volvox aureus, Volvox carteri

, and

H. plusialis

and

Platymonas subcordiformis

(a prasinophycean) and

Gym-

nodinium splendens

(a dinophycean) (Foster et al. 1984).Reconstitution of phototaxis with retinal in

C. rein-hardtii

cells that are blind with respect to photomove-ment supplied convincing evidence that the photore-ceptor is rhodopsin (Foster et al. 1984). Then, therhodopsin chromophore was characterized

in vivo

ex-tensively by supplementation of white cells with a vari-ety of retinal analogs and studying the behavior andthe photoreceptor currents by several different meth-ods. In addition, retinal was extracted from wild-typecells and analysed. The conclusion was that the algalrhodopsin(s) contains all-

trans

retinal that isomerizesin light to 13-

cis

similarly to archaean rhodopsin (type1 rhodopsins) but differently from animal rhodopsinwhere the 11-

cis

retinal isomerizes in light to all-

trans

(type 2 rhodopsins) (reviewed by Spudich et al. 1995,Hegemann 1997, Sineshchekov and Govorunova 1999).

Supplementation of white retinal-deficient cells with

3

H-retinal or exchanging the endogenous retinal in pu-rified eyespot membranes against

3

H-retinal identifiedonly one single retinal binding protein, which has beenpurified and sequenced (Deininger et al. 1995). Becauseof its sequence homology to invertebrate opsins (type 2opsins) it was named chlamyopsin. But chlamyopsin(Cop) as well as its homolog from

Volvox carteri

, volvox-opsin (Vop, Ebnet et al. 1999) are highly charged andthe overall sequences are hardly compatible with a 7TM-receptor. In addition, the sequences were hardly compa-rable to the type 1 rhodopsin chromophore character-ized

in vivo.

It was proposed from electrophysiologicaldata that algal opsins form a complex with the ionchannel protein constituting the high light-saturatingion conductance responsible for the photophobic re-sponses (Holland et al. 1996). Furthermore, antiseraagainst chlamyopsin impair the light-regulated GTPaseactivity of eye-specific G-proteins, suggesting that aG-protein is involved in the transduction process(Calenberg et al. 1998) as it is in vision of higher ani-mals. Although the identified algal opsins are thedominant proteins of the eye (Deininger et al. 1995,Fuhrmann et al. 1999), they may also be expressed inother regions of the cell. For example, volvoxopsin is

expressed in eyeless gonidia and young embryos, longbefore the cells are fully differentiated and pig-mented eyes are formed. Volvoxrhodopsin may have asecond function besides its suggested involvement inphotomovement responses (Ebnet et al. 1999).

A genetic analysis of the opsin function has beenunsuccessful thus far because opsin mutants are notavailable and targeted gene disruption has yet to beestablished for any green alga. To overcome themethodological problems that prevent disruption ofgenes in green algae, an antisense-RNA approach wastried in

Volvox carteri

(Ebnet et al. 1999). Simply,gonidia were transformed with a volvoxopsin genewith partially inverted regions (vop). Transformantswith multicopy gene integrations and a reduced opsinlevel were found, that showed a reduction of theirphototactic sensitivity. The transformants, however,were unstable, and photocurrent measurements werenot possible due to the vast amount of extracellularmatrix. Later, a more efficient antisense approach wasapplied to C. reinhardtii. Intron-containing gene frag-ments directly linked to their intron-less cDNA anti-sense counterpart provide efficient post-transcrip-tional gene silencing (PTGS) thus allowing efficientreduction of a specific gene product in C. reinhardtii(Fuhrmann et al. 2001). In the opsin-deprived trans-formants, the photoreceptor currents, the photopho-bic response, and phototaxis were left unchanged.This led to the conclusion that the identified opsin-related proteins (type 2 opsins) are not the photore-ceptors for photophobic responses in C. reinhardtii.These long-studied responses must be triggered by anovel, so far unidentified, rhodopsin species.

The only alga in which a second retinal protein hasbeen identified is Dunaliella salina (Fig. 2a). This wasnot surprising because phototaxis and photophobicresponses exhibit rhodopsin action spectra with dif-ferent maxima (Wayne et al. 1991). More specifically,labeling of eyespot membranes with 3H-retinal identi-fies a 28 kDa retinal-binding protein, probably of thechlamyopsin and volvoxopsin type. In addition, a largerretinal protein of 45kDa was identified in this alga (Fig.2a) suggesting that a second class of rhodopsins mightexist in green algae. Recently, cDNA-sequences appearedfrom the C. reinhardtii genome project, which code for a56 kDa opsin type I protein (AccNo AF385748). This pro-tein shows homology to the sensory rhodopsins from thearchaea (�21%) as well as to the rhodopsin from Neu-rospora (NOP1, �14%), the function of which is stillunclear. The homology might appear small, but mostamino acids that define the retinal binding site andthe H�-conducting ion channel are conserved (Fig.2b). In addition, the topology with 7 transmembranesegments (Fig. 2b) and the hypothetical retinal-bind-ing site (Fig. 2c) suggests that this protein is an excel-lent candidate for the C. reinhardtii homologue of the45 kDa retinal protein of D. salina and is the photore-ceptor for phobic responses and phototaxis in C. rein-hardtii. This protein, as well as its rhodopsin counterpartfrom Neurospora, might function as a light-driven ion

ALGAL SENSORY PHOTORECEPTORS 673

channel. If this hypothetical role can be confirmed, thengreen algae contain both type 1 and type 2 rhodopsins.

phototaxis in euglenaAnother alga in which photomovement has been

studied for decades is Euglena gracilis (Euglenophyta).The clearest action spectrum in the literature for pho-tophobic responses and phototaxis of Euglena was re-corded by B. Diehn in 1969. Both are clean flavin spec-tra with maxima at 370 and 470 nm. The light sensitiveorganelle is the paraflagellar swelling (PFS), previouslynamed the paraflagellar body (PFB). The PFS is lo-cated at the base of the long flagellum within the am-pulla of the alga. It is made up of a three-dimensionalcrystal of photoreceptor protein, which provides direc-tional sensitivity of the photoreceptor due to this highlyordered arrangement (Piccini and Mammi 1978). Thephotoreceptor becomes highly fluorescent under ex-tended radiation with 370 nm light, and the fluores-cence rapidly decays in 450 nm light (Barsanti et al.1997). These are typical characteristics of a flavin-basedphotoreceptor. The flavin nature of the receptor wassupported in blind E. gracilis strains that have low fla-vin content. Phototaxis was reconstituted by additionof riboflavin and roseoflavin, the latter shifting the ac-tion spectrum to longer wavelength (Häder and Leb-ert 1998). However, the flavin nature of the photore-ceptor is not generally accepted. Gualtieri et al.(1992) extracted retinal from whole organisms andused the presence of retinal as evidence supportingrhodopsin as the photoreceptor. Moreover, E. graciliswas incubated for several days in hydroxylamine, a clas-

sical reagent for the extraction of retinal from rhodop-sins. After this treatment, E. gracilis was not phototactic,but, because of the general oxidative character of thereagent, the cells remained much smaller than theiruntreated relatives (Barsanti et al. 1993). Summarizingthe pros and cons in favor of a flavin- or a rhodopsin-based photoreceptor, a shift of the action spectrumupon application of a modified flavonoid is the clear-est available argument in favor of a flavin-based pho-toreceptor, but the question is far from being solved.This natural crystal is one of the most attractive sys-tems for any kind of biophysical study.

rhythmicityAlgae, like most other organisms, do not merely re-

spond to their environment; they also have the capac-ity to adjust their physiology and behavior in anticipa-tion of changing environmental conditions (Roennebergand Foster 1997). Lower eukaryotes, especially, are ableto acclimate only to environmental changes that happenperiodically. The major rhythmic change is the day/night cycle. The machinery that prepares an organismfor the day or the night is the biological or circadianclock. The timing of the clock, however, is not perfectand only is circa a day and it needs an input signalfrom a Zeitgeber to be precisely in time. The mechanismof the clock can be part of the molecular biology of asingle cell, as demonstrated for a variety of prokaryoticand eukaryotic algae such as Synechococcus, Acetabularia,Chlamydomonas, Euglena, and Gonyaulax ( Johnson andSuzuki 2001, Mittag 2001 and literature cited therein).As in Chlamydomonas, the Gonyaulax circadian system

Fig. 2. (a) Retinal-binding proteins of C. reinhardtii and Dunaliella salina eyes. Eyespot membranes were incubated with 3H-retinaland subsequently reduced with NaBCNH3 according to Deininger et al. (1995) and separated by SDS-gel electrophoresis. (b) 3D-backbone model of the hypothetical type 1 rhodopsin C. reinhardtii encoded by the cDNA: AccNo AF385748. The deduced amino acidsequence was aligned and modeled to the BR-structure 1FBB (Subramaniam and Henderson 2000) from PDB (Berman et al. 2000),as a template using SWISS-MODEL and Swiss PDB-viewer 3.7b2 available from http://www.expasy.ch/swissmod/SWISS-MODEL.htmlwith default parameters. Note that the loop structure, the end of helix 4, and the extended N and C termini are not completely repre-sented. (c) A section through a space filling model of the same protein showing the retinal in yellow and amino acids identical to thereference bacteriorhodopsin from Halobacterium salinarium in cyan. Cop � chlamyopsin; Dop � dunaliellaopsin.

674 PETER HEGEMANN ET AL.

responds to light both in the blue and the red part ofthe spectrum (475 and 650 nm). The first clear indica-tion for the involvement of more than one photorecep-tor originates from experiments in which the period’sdependence on the light’s fluence rate (period-inten-sity relationship) was measured in monochromatic light.Increasing fluence rates of blue light shortened the pe-riod, whereas a higher photon irradiance of red lightlengthened it (Roenneberg and Hastings 1988). Thetwo photoreceptors involved remain to be identified.

Rhythmic circadian growth of Porphyra umbilicalis(Rhodophyta) was studied under different spectrallight conditions, and the phase shift response curveshave been recorded for blue green and red light. Thehigh efficiency of blue light in shifting the growthrhythm is similar to rhythm entrainment in Arabidop-sis, which led to the suggestion that a Cry1-like photo-receptor is present in P. umbilicatis (Lüning 2001 andliterature cited therein).

LIGHT-INDUCED DEPOLARISATION IN ACETABULARIA

The alga Acetabularia acetabulum (formerly called A.mediterranea) has a cell volume that is large enough forperforming intracellular voltage clamp experiments.A step-up stimulation of the alga with blue and redlight results in hyperpolarization of the plasmalemma,which is a result of photosynthesis. Schilde (1968) no-ticed that a rapid green-light-induced depolarizationpreceded the hyperpolarization. The action spectrumof the depolarization is rhodopsin-shaped, and Schildewas the first to postulate the presence of a rhodopsinin a plant system (Schilde 1968). Later Gradmann ex-plained the depolarization as an inhibition of the elec-trogenic chloride pump by a permeability increase ofthe pump for the carrier ion (Gradmann 1978). Anti-bodies against chlamyrhodopsin-1 and 2 identified arelated protein in Acetabularia; however, this protein ispresent only in the small, motile gametes, which havenot been subjected to electrophysiological studies yet.The gamete opsin is not present in the mature benthicalga (Hegemann and Gradmann, unpublished). Thismeans that the photoreceptor responsible for the rapiddepolarization of mature cells also remains to be iden-tified. As in freshwater algae, more than one rhodop-sin-like photoreceptor is expected to operate in theDasycladales.

OUTLOOK

The classic way to identify the nature of a photore-ceptor was and still is action spectroscopy. Clear ac-tion spectra, however, are only obtained if basic physi-cal principles, as they were carefully developed by M.Delbrück and colleagues in the late 1960s, are takeninto account (Shropshire 1972, a broad discussionabout action spectroscopy is given by Foster 2001b).Threshold action spectra are generally more accuratethan equal response spectra because threshold spec-tra eliminate adaptation phenomena and modulation

of the screening (Foster 2001b). Moreover, flash re-sponses result in better action spectra than responsesrecorded in continuous light, if the responses toflashes are visible clearly enough to allow quantifica-tion, for example by tracking individual cells underthe microscope. For the identification of rhodopsin ingreen algae, the breakthrough was reached after re-constitution of blind retinal-deficient cells with elec-tronically-altered retinal analogs. The shifted actionspectrum was the clearest physiological indication fora retinal-based photoreceptor. Retinoids should beadded at low concentration, however, to avoid recon-stitution of the receptor with minor contaminants,which originally lead to a misinterpretation of the al-gal rhodopsin as an animal-type rhodopsin with a 11-cis retinal chromophore (Foster et al. 1984).

In the case of flavin receptors, this approach wasnot successful, either in algae or in higher plants, forseveral reasons: flavin-deficient mutants do not existbecause flavin-deficiency is lethal; pure flavins cannotbe metabolized to riboflavin, FMN or FAD�; flavinbinds orders of magnitude less tightly to the apopro-teins than riboflavin, FMN or FAD; finally, in pure fla-vin containing an N10-H bond, the isoalloxacine ringisomerizes to alloxacine, which is much less reactivethan isoalloxacine. Therefore, analogs must be addedas riboflavin derivatives to compete efficiently with theendogenous FMN or FAD�. This should be taken intoaccount for further studies. Finally, flavin-based pho-toreceptors may be active with the FMN or FAD� fullyoxidized, fully reduced, or as semichinone radicals. Ac-cordingly, the absorption maxima are expected to be at390, 460 or between 600 and 700 nm, and the extinc-tion coefficient varies between 1000 to 15000 M1cm1.Due to these difficulties, the breakthrough for the iden-tification of cryptochromes and phototropins camein higher plants from photoreceptor mutants, whichwere generated long before the receptors were identi-fied. Thus, generation of algal mutants with clear phe-notypes is urgently needed. In case of macroalgae, themaintenance of many mutants is difficult, but, in thenear future potential algal photoreceptor genes willappear from genome sequencing projects, as alreadyexemplified for C. reinhardtii. To prove the function,these genes have to be inactivated. The establishmentof a method for site-directed gene replacement ormodification is, therefore, also needed, but to myknowledge targeted gene disruption is not possible inany alga (except prokaryotic blue-green algae).

Finally, it should not be overlooked that the func-tion of algal photoreceptors might be quite differentfrom their function in higher animals or higher plants.For instance, rhodopsins function as G-protein activa-tors in animals as a H�-pump, Cl pump, or activatorof His-kinase cascades in archaea, and probably as light-gated ion channels in algae. Likewise, critical studiesand elucidation of blue-light receptors are still neededto define phenomena such as the fucoid blue-light-induced polarization of zygotes (already carefully de-scribed by Hurd in 1925).

ALGAL SENSORY PHOTORECEPTORS 675

Only the combination of modern gene technologytogether with profound physiology and highly sophis-ticated biophysical studies of over-expressed receptorswill provide a full understanding of photoreceptorfunction.

We thank Drs. C. Beck, N. A. Reisdorph and G. Small for shar-ing unpublished observations with us. We also thank Drs. C.Beck, S. H. Brawley, D. Gradmann, and G. Wagner for manyhelpful comments.

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