- Email: [email protected]
Rhodopsin-mediated photosensing in green flagellated algae
trends in plant science reviews 33 Bol, J.F. et al. (1996) Regulation of the expression of plant defense genes, Plant Growth Regul. 18, 87–91 34 Peña-Cortés, H. et al. (1993) Aspirin prevents wound-induced gene expression in tomato leaves by blocking jasmonic acid biosynthesis, Planta 191, 123–128 35 Doares, S.H. et al. (1995) Salicylic acid inhibits synthesis of proteinase inhibitors in tomato leaves induced by systemin and jasmonic acid, Plant Physiol. 108, 1741–1746 36 Niki, T. et al. (1998) Antagonistic effect of salicylic acid and jasmonic acid on the expression of pathogenesis-related (PR) protein genes in wounded mature tobacco leaves, Plant Cell Physiol. 39, 500–507 37 Lawton, K.A. et al. (1994) Acquired resistance signal transduction in Arabidopsis is ethylene independent, Plant Cell 6, 581–588 38 van Loon, L.C., Bakker, P.A.H.M. and Pieterse, C.M.J. (1998) Systemic resistance induced by rhizosphere bacteria, Annu. Rev. Phytopathol. 36, 453–483 39 Bakker, P.A.H.M., Van Peer, R. and Schippers, B. (1991) Suppression of soilborne plant pathogens by fluorescent Pseudomonads: mechanisms and prospects, in Biotic Interactions and Soil-Borne Diseases (Beemster, A.B.R. et al., eds), pp. 217–230, Elsevier 40 De Meyer, G. and Höfte, M. (1997) Salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 induces resistance to leaf infection by Botrytis cinerea on bean, Phytopathology 87, 588–593 41 Maurhofer, M. et al. (1998) Salicylic acid biosynthetic genes expressed in Pseudomonas fluorescens P3 improve the induction of systemic resistance in tobacco against tobacco necrosis virus, Phytopathology 88, 678–684 42 Pieterse, C.M.J. et al. (1996) Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression, Plant Cell 8, 1225–1237 43 Van Wees, S.C.M. et al. (1997) Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria, Mol. Plant–Microbe Interact. 10, 716–724 44 Hoffland, E. et al. (1995) Induced systemic resistance in radish is not associated with accumulation of pathogenesis-related proteins, Physiol. Mol. Plant Pathol. 46, 309–320
45 Press, C.M. et al. (1997) Salicylic acid produced by Serratia marcescens 90-166 is not the primary determinant of induced systemic resistance in cucumber or tobacco, Mol. Plant–Microbe Interact. 10, 761–768 46 Pieterse, C.M.J. et al. (1998) A novel signaling pathway controlling induced systemic resistance in Arabidopsis, Plant Cell 10, 1571–1580 47 Ryals, J.A. et al. (1997) The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor IkB, Plant Cell 9, 425–439 48 Cao, H. et al. (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats, Cell 88, 57–63 49 Knoester, M. et al. (1998) Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi, Proc. Natl. Acad. Sci. U. S. A. 95, 1933–1937 50 McConn, M. et al. (1997) Jasmonate is essential for insect defense in Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 94, 5473–5477 51 Staswick, P.E., Yuen, G.Y. and Lehman, C.C. (1998) Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare, Plant J. 15, 747–745 52 Vijayan, P. et al. (1998) A role for jasmonate in pathogen defense of Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 95, 7209–7214 53 Thomma, B.P.H.J. et al. (1998) Separate jasmonate-dependent and salicylatedependent defense response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens, Proc. Natl. Acad. Sci. U. S. A. 95, 15107–15111
Corné M.J. Pieterse* and Leendert C. van Loon are at the Section of Plant Pathology, Dept of Plant Ecology and Evolutionary Biology, Utrecht University, PO Box 800.84, 3508 TB Utrecht, The Netherlands.
*Author for correspondence (tel 131 30 253 6887; fax 131 30 251 8366; e-mail [email protected]; internet http://www.bio.uu.nl/~fytopath).
Rhodopsin-mediated photosensing in green flagellated algae Oleg A. Sineshchekov and Elena G. Govorunova Green flagellated algae possess a primitive visual system that regulates the activity of their motor apparatus. Photoexcitation of a rhodopsin-type photoreceptor protein gives rise to the photoreceptor current, which, above a certain threshold of stimulus intensity, induces the flagellar current. It is probable that the photoinduced alteration in flagellar beating is governed by changes in intracellular Ca21 concentration. This rhodopsin-mediated sensory system serves to align the swimming path with the direction of the light stimulus, whereas processes of energy metabolism determine whether the oriented movement is directed towards or away from the light source.
S
urvival of phototrophic organisms depends on their ability to optimize their exposure to sunlight. Light controlled cell motility appeared early in evolution as one of the strategies used to achieve this goal. Unicellular flagellated algae, as well as motile zoospores and gametes of macroalgae, actively search for optimal light conditions by means of phototaxis (i.e. oriented swimming towards or away from the light source)1,2. In addition, a sudden change in light intensity, irrespective of its direction, usually elicits a photophobic or photoshock cell response, which
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appears as a transient stop, often followed by a brief period of backward motion1. Most flagellates rotate around their longitudinal axis during forward swimming. The ability to track the direction of light is based on the capacity to sense a temporal difference in the amount of quanta captured in a single photoreceptive region of the cell under lateral illumination. Structural and functional aspects of phototactic sensory systems found in different taxonomic groups of algae are very diverse2 and might reflect their independent evolutionary origin. Chlorophyceae are a unique group of
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trends in plant science reviews microorganisms, because the photosensory transduction mechanisms found in these algae are strikingly similar to the processes of animal vision2,3. Therefore, elucidation of these mechanisms might give a new insight into the evolution of photosensory systems. Photoreceptor apparatus
Light
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Unbalanced flagellar response (phototaxis)
Eyespot PC 2 Green flagellated algae usually carry an 221111 2 FC Rhodopsin PC 12 12 eyespot, seen under the light microscope as 12 photoexcitation generation 2 1111 2 a highly pigmented organelle from about 22 2 2 one to a few mm in size. The eyespot is Electrotonic often located at the cell equator within the spread of membrane chloroplast close to the cell surface, and is depolarization FC Flagellar displaced by 20–408 from the plane of flageneration undulation gellar beating. The precise position of the (photophobic eyespot is probably ensured by its strucresponse) tural association with the flagellar root sysFig. 1. Rhodopsin-mediated signal transduction chains for phototaxis and photophobic tem4. The eyespot consists of one to several response in green flagellated algae. Photoexcitation of the photoreceptor rhodopsin leads to layers of carotenoid globules, often subgeneration of the photoreceptor current (PC) across the eyespot region of the cell memtended by specialized thylakoid membrane. PC generation is responsible for phototaxis, which appears to be based on differen4,5 branes . The visible pigment is, however, tial sensitivity of the axonemes to Ca21. The molecular mechanism of the coupling between not the photoreceptor pigment, which is the PC generation and the unbalanced flagellar response has yet to be established. When present in a much lower concentration and light stimulus intensity exceeds the critical level necessary to evoke the photophobic response, a flagellar current (FC) is observed. Ca21 channels in the flagellar membrane are is assumed to be embedded in the plasma activated by the PC-induced depolarization, which electrotonically spreads over the whole membrane and/or the outer chloroplast enplasma membrane. Changes in the membrane potential, induced by generation of the PC velope membrane in the area overlaying 4 and the FC, can so far only be indirectly estimated. Therefore the distribution of the positive the eyespot . The photoreceptor membranes and negative charges shown here corresponds to the direction of the change (i.e. the resting and the eyespot together form the photorepotential becomes less negative), and not to the absolute value of the potential upon photoceptor apparatus. The importance of the excitation. The FC is carried by a massive influx of Ca21 ions, which leads to conversion of eyespot for normal phototactic orientation flagellar beating mode from ‘breast stroke’ to undulation. has been demonstrated in experiments with an eyespot-deficient Chlamydomonas mutant6. When the photoreceptor apparatus is turned away from the light source, photoreceptor illumination is and a ‘dissolver’ Volvox mutant16, whereas the suspension method attenuated by absorption and scattering of light by the eyespot. In of photoelectric measurements is more universal. Common feaaddition, multi-layered eyespots, present in many species of green tures of the photoelectric responses found in different species sugflagellates, appear to be quarter-wave interference reflectors, gest that the same basic scheme of sensory transduction holds for which produce maximum light intensity at the presumed photo- the whole group of chlorophyceae (Fig. 1). receptor site when the photoreceptor apparatus is facing the light The earliest detectable event in the signal transduction chain stimulus5,7. Illumination of the photoreceptor does not change dur- for both phototaxis and photophobic response is the generation of ing the rotation cycle when the cell swims parallel to the light di- an inward current across the portion of the plasma membrane rection, but becomes periodic when the cell deviates from it. This overlaying the eyespot, defined as the photoreceptor current (PC; periodic signal is perceived as a stimulus for correcting the swim- Refs 13,15). Switching on the light induces a transient PC peak ming path5. Light absorption by the bulk chloroplast pigments also which decays to a lower stationary level and dissipates after contributes to modulation of the photoreceptor illumination6,8. switching off the light (Fig. 2). The action spectrum for PC genFinally, preferential absorption (dichroism) of the photoreceptor eration is very close to the action spectra for photomotile cell itself, resulting from the highly ordered orientation of the chromo- responses. The role of the PC in phototaxis was revealed by phores9,10, probably adds to the directivity of the photoreceptor correlation of electrical responses to photoinduced changes in apparatus5. flagellar beating observed in a cell held on a pipette. Step-up stimulus induced an increase in the beat frequency of the cisSignal transduction mechanisms flagellum (the one closest to the eyespot), and a decrease in that of Electrical processes play a key role in photosensory transduction the trans-flagellum, whereas step-off stimulus caused the oppoin green flagellated algae9,11,12. Algal cells do not usually exceed site effect9,11. Photoinduced changes in front amplitude, which 10–20 mm in diameter, which makes microelectrode recording were opposite in the cell’s two flagella, were also reported9,11,17. rather problematic. Instead, the photoinduced electrical signals Such unbalanced motor responses of the two flagella would lead involved in phototaxis and photophobic response in green flagel- to the correction of the swimming path in a freely swimming cell lates can be measured extracellularly from individual cells13 and (i.e. to phototaxis). from cell suspensions14. Extracellular recording takes advantage Complex kinetics of the PC (Ref. 18) and the biphasic deof the asymmetric localization of the signal sources within the pendence of its peak amplitude on stimulus intensity11,14,19 led to cell. Application of the suction pipette technique is limited to rela- the conclusion that two different electrical processes contribute tively large flagellates with elastic cell walls such as wild-type to PC generation9,11. Upon flash excitation, the first process deHaematococcus13, cell wall-deficient Chlamydomonas mutants15 velops within the time resolution of the measuring system (,5 ms; February 1999, Vol. 4, No. 2
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Light off
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Fig. 2. Photoelectric responses elicited in the same cell of Haematococcus pluvialis by a pulse of continuous light at two different fluence rates:1, 7.5 3 1019 photons m22 s21; 2, 1.75 3 1019 photons m22 s21. FC; flagellar current: PC; photoreceptor current.
Fig. 3). Its peak amplitude saturates at extremely high light intensities, indicating that it is limited only by photoconversion of the photoreceptor protein11. The delay time of the second process increases with the decrease in flash intensity and could extend to a few ms (Fig. 3), indicating the involvement of intermediate biochemical steps in signal transduction. Light saturation of its peak amplitude can be up to three orders of magnitude lower than that of the first process, and its maximum peak amplitude reaches only about 10% of that of the first process (Fig. 3). The two processes that contribute to PC generation were defined, according to their features, as the ‘early PC’ and the ‘late PC’ (or, alternatively, as the ‘high-saturating PC’ and the ‘low-saturating PC’). At high stimulus intensity, the recorded PC mostly consists of the early PC, whereas at low stimulus intensity, the late PC makes a major contribution to the recorded signal. Especially long delay times for PC at low stimulus intensity have been recently reported in Volvox16. At high stimulus intensity, the number of elementary charges transported across the photoreceptor membrane (calculated from the area under the curve of PC) is close to the number of photons absorbed by the photoreceptor. Taking the virtually instant onset of the early PC into account, there are two possibilities, either the early PC results from translocation of the ions across the membrane by a low-conductance ion channel closely associated with the rhodopsin12, or even by the rhodopsin itself. At physiological conditions PC is mostly driven by the influx of Ca21 ions13,15, although a Ca21-independent component is also present13,20, which in Volvox has been defined as a ‘slow PC’ (Ref. 16). A drop in the external pH dramatically increases PC even in the absence of external Ca21, which points to the contribution of H1 influx to PC generation, rather than to acid facilitated Ca21 influx16. The amplitude of the stationary current, which is probably determined by the late PC, corresponds to about 107 elementary charges per s being transported across the membrane when 103 quanta per s are absorbed by the photoreceptor9,11. This means that generation 60
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High-saturating early PC
4 Peak current (nA)
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Fig. 3. (a) Photoelectric responses evoked in a cell suspension of Chlamydomonas reinhardtii strain 495 (1) at different flash intensities. Photo-flash excitation, relative stimulus intensity: 1, 100%; 2, 2.5%; 3, 0.01%. Arrows indicate the time of the flash. Broken line shows the response delay in trace 3. Inset: the onset of the photoreceptor current (PC) recorded with an improved time resolution in Chlamydomonas reinhardtii strain 516/white-3 supplemented with 1028 M all-trans retinal. Laser flash excitation, 500 nm, 2 J m22. The laser artifact is digitally subtracted. (b) Dependence of the PC peak amplitude measured in a cell suspension of Chlamydomonas reinhardtii strain 495 (1) on the fluence of the excitation flash. Solid line shows a computer fit to Michaelis function for the late PC and to exponential function for the early PC.
of the late PC involves up to four orders of signal amplification. The basis of the amplification cascade involved in generating the late PC is not yet known, but recent biochemical and immunological studies on isolated photoreceptor apparatuses point to the presence of G proteins, which are also involved in the enzymatic cascade of animal photosensory transduction21. Most convincingly, light-dependent GTPase activity, with an action spectrum similar to that of rhodopsin absorption, was found in isolated eyespot apparatuses of the green alga Spermatozopsis, which was inhibited by antibodies raised against Chlamydomonas rhodopsin22. In addition, Ca21-dependent protein kinase and phosphatase activities were found in a similar in vitro preparation21,23. Experiments in reactivated, demembranated Chlamydomonas cell models revealed different sensitivities of cis- and trans-axonemes
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Direction
Sensory transduction
Modulation of photoreceptor illumination
Unbalanced flagellar response
Degree of orientation Cell alignment
Light
Cell orientation Movement along the intensity gradient
Intensity gradient
to Ca21 concentration, which might account for the unbalanced motor response of the two flagella, which is necessary for phototaxis1. This Ca21-dependent shift in flagellar dominance was not found in the cell models of a non-phototactic ptx1 mutant that lacks a pair of 75-kDa axonemal proteins24. In this mutant, photoinduced changes in beat frequency and amplitude occur in both flagella as in the trans-flagellum in the wild type25. The ptx5, ptx6 and ptx7 mutants probably have other defects in genes encoding axonemal proteins responsible for this shift26. Asymmetric phosphorylation of a 138-kDa protein of an inner dynein arm complex appears to be one more factor to determine different sensitivity of the two axonemes to Ca21 associated with phototaxis27. When the intensity of the light stimulus exceeds a certain threshold, PC becomes superimposed by an ‘all-or-nothing’ spikelike response (Fig. 2). The increase in stimulus intensity shortens its delay time from over 60 down to 5 ms (Refs 13,15). It reflects an inward current across the flagellar membrane and is therefore defined as the ‘flagellar current’ (FC; Ref. 15). No FC could be recorded immediately after complete mechanical amputation of flagella, and the time course of the signal recovery was found to correlate with that of flagellar regrowth28. The area under the curve of PC before the beginning of the FC is nearly constant over a wide range of stimulus intensities9,11,12. This indicates that a certain amount of charge should enter the cell to initiate the FC. When the cell is hyperpolarized by photosynthetically-active red background illumination, this integral increases twofold9,11. Therefore it could be concluded that FC is activated by the PC-induced depolarization of the membrane to a certain level. FC is more sensitive to the removal of Ca21 from external medium than PC (Ref. 13), and is most probably driven by the activation of voltage-gated Ca21-channels in the flagellar membrane15,28. FC can also be observed at low external pH in Ca21-free medium, which indicates that flagellar channels are partially permeable to protons29. They are likely to be perturbed in ppr1–ppr4 mutations in Chlamydomonas30, whereas ptx2 and ptx8 strains probably lack an element in the signal transduction chain, which is located downstream from PC generation and is common for the phototaxis and the photophobic response pathways26. FC drives a switch from ‘breast-stroke’ flagellar beating to flagellar undulation, which is characteristic of the photophobic response in a freely swimming cell. This notion has been proven by simultaneous recording of photocurrents and flagellar beating from the same cell held on a micropipette31. No spike-like FC was found in Volvox, the cells of which react to photophobic stimuli only by a transient arrest of flagellar beating rather than by a switch to flagellar undulation16. Externally applied electrical depolarization activates flagellar Ca21 channels and induces conversion of beating mode in Chlamydomonas flagella32. Spontaneous spikes similar to FC can sometimes be observed in the dark or under continuous illumination13,31, which might play a role in regulation of spontaneous directional changes (klinokinesis). Studies on isolated, reactivated flagellar apparatuses and axonemes have established that Ca21 acts directly on the axoneme and at around 1024 M induces a switch from ciliary type stroke to undulation, probably involving Ca21-dependent phosphorylation of specific axonemal proteins1–3. The spike-like FC, also called the ‘fast flagellar current’ (Ff), is accompanied by the ‘slow flagellar current’ (Fs) with a peak time of a few hundred ms12, the amplitude of which dramatically increases upon substitution of external Ca21 for Ba21 (Ref. 20). The functional role of Fs is presumably related to controlling the duration of backward swimming during the photophobic response12,20.
Control of the response sign
Sign of phototaxis Cell illumination level
Energetic state of cell
Photosynthesis
Fig. 4. Two feedback loops of light controlled behaviour in green flagellated algae. Upper circle: a rhodopsin-mediated sensory system is responsible for the alignment of the swimming path with respect to the light stimulus. Tracking the light direction is based on modulation of the photoreceptor illumination during the rotation cycle. Deviation of the swimming path from the stimulus direction induces a change in the pattern of photoreceptor illumination, which initiates a corrective motile response. Lower circle: absorption of the light by photosynthetic apparatus determines the energy level of the cell, which controls the resting membrane potential. The resting membrane potential is involved in regulation of the sign of phototaxis (swimming towards or away from the light source). The net behavioural response results from a combination of the rhodopsin-based and photosynthesis-driven regulatory processes and leads to an accumulation of the cells in the area of optimal illumination.
Generation of Fs and stationary PC seems to be linked to K1 efflux, which counterbalances depolarization of the membrane caused by these long-lasting inward currents16,29. Transient K1 currents are triggered by membrane depolarization induced by Ff (Ref. 33); their direction can be inward or outward depending on the K1 electrochemical driving force. Photosensory adaptation
The phototaxis response does not become desensitized upon repetitive light stimulation, which leads to prominent desensitization of the photophobic response34. Precise desensitization of the photophobic response at high stimulus intensities provides the large dynamic range of phototaxis35. Desensitization and dark recovery of the cell upon photoexcitation is determined by changes in the membrane potential33. FC, which is only necessary for the photophobic response, causes stronger depolarization of the plasma membrane than PC alone, and consequently increases the refractory period upon light stimulation. One of the recovery mechanisms involved in restoration of the resting membrane potential after depolarization induced by FC, is an outward K1 current observed at low external K1 concentrations33. February 1999, Vol. 4, No. 2
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trends in plant science reviews Photoreceptor protein
Initial information on the nature of the photoreceptor responsible for phototaxis in green flagellated algae has been obtained by action spectroscopy. The maximum efficiency of wavelengths of around 500 nm for most green flagellates favoured the view that carotinoproteins were the photoreceptor pigments. The hypothesis has been suggested that these proteins might resemble rhodopsins involved in animal vision5. This has been confirmed by experiments in which phototaxis in blind carotenoid-deficient Chlamydomonas mutants was restored upon the addition of exogenous retinal and its analogues36. Reconstitution studies undertaken with a variety of retinoid compounds, discussed in detail in a recent review37, led to the conclusion that all-trans, 6-s-trans retinal is the native chromophore in the Chlamydomonas rhodopsin as in archaebacterial rhodopsins. Photoexcitation gives rise to its isomerization to a 13-cis form, which again resembles archaebacterial rhodopsins, whereas the primary event in animal vision is isomerization of 11-cis-retinal to all-trans-retinal. The functional Chlamydomonas photoreceptor requires the presence of at least three conjugated double bonds and the 13-methyl group in the polyene chain of its chromophore. The addition of all-transretinal, 9-dimethyl-retinal and dimethyl-octatrienal results in the appearance of the normal PC, which proves that restoration of behavioural responses is indeed due to reconstitution of the functional photoreceptor19. Hydroxylamine, the agent known to induce light-dependent cleavage of the chromophore in retinal-containing proteins, causes a specific inhibition of phototaxis and PC in wildtype cells3,19. These in vivo results have been complemented by HPLC detection of retinoids in cell extracts2,3. A single 30-kDa protein was identified in Chlamydomonas carotenoid-deficient cells by 3Hretinal labelling38. Application of a similar procedure to the eyespot fraction isolated from wild-type cells allowed purification of the presumed photoreceptor protein to electrophoretic homogeneity38. Polyclonal antibodies raised against Chlamydomonas opsin concentrated precisely in the eyespot area in permeabilized cells thus indicating localization of the rhodopsin in vivo38. The opsin cDNA was purified and sequenced38, and genomic opsin clones were isolated from Chlamydomonas and Volvox3. The derived opsin amino acid sequences are 65% identical and show no homology to bacterial opsins, in spite of the similarity in chromophore properties described above. On the other hand, some homology to animal opsins, especially those from invertebrates, was apparent. Algal opsin sequences are enriched in polar amino acid residues and bear some homology with ion channel sequences38, which corroborates the above hypothesis that the rhodopsin itself might form the light-regulated membrane conductance. Regulation of phototaxis by photosynthesis and other processes of energy metabolism
Phototaxis in green flagellated algae is mediated by a specific photoreceptor system that is separate from the photosynthetic apparatus. However, cell accumulation in the area of optimal light conditions for phototrophic metabolism requires the involvement of a negative feedback loop in light control of photomovement. This loop is provided by regulation of phototaxis by photosynthesis and other energy conversion processes (Fig. 4). A rapid switch from positive to negative phototaxis (swimming towards, or away from the light source, respectively) can be induced by a red background illumination that is by itself insufficient for photoorientation19,39. Red light induces hyperpolarization of the plasma membrane and the increase in PC amplitude18, which leads to the reversal of phototaxis sign. Spontaneous changes of swimming direction about every 25–30 s were observed both in dark and at 62
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moderate stimulus intensities in Chlamydomonas and Haematococcus40. This behaviour is sensitive to red light, and is probably related to the periodic electrical processes in the plasma membrane detected by extracellular microelectrodes9. A phase shift between the two periodic electrical processes, presumably associated with the two flagella of the cell observed upon turning on the light might control the switch from positive to negative phototaxis. Prospects
Although a general overview of the signalling pathways involved in light control of behaviour in green flagellated algae has been outlined in recent years, identification and characterization of individual molecular components of the photosensory system has only just begun. Detailed genetic analysis and phenotypic characterization of mutants defective in phototaxis and photophobic response should help to dissect the rhodopsin-mediated signal transduction chain. Sequencing opsins from Chlamydomonas and Volvox provides the basis for genetic engineering of the key elements of this chain. Overexpression of opsins and other gene products involved in photosensory transduction in green flagellates is probably the only way in which to collect enough protein for spectroscopic and biochemical studies. The physiological significance of individual components of the signalling pathway should be verified by investigating photosensory function in vivo by means of non-invasive physiological techniques such as photoelectric measurements in cell suspensions. Physiological studies on intact cells would also help to elucidate how the photosensory system is integrated in general cell metabolism. By extending the research to a wider range of species it might be possible to identify subjects that are more amenable for comparative analysis and suitable for modern experimental techniques, such as patch clamping and the use of ion-specific fluorescent indicators. Comparative analysis of signal transduction mechanisms in different species of green flagellates is highly desirable for understanding the evolution of rhodopsin-mediated signalling systems. Acknowledgements
We would like to thank Dr F-J. Braun and Prof. P. Hegemann for providing us with their manuscript submitted for publication, and Dr G. Kreimer, Dr U. Rueffer and Prof. W. Nultsch for the reprints of their recent work. We thank I.M. Altschuler for his help in preparation of the manuscript. This work was supported by INTAS-RFBR grant No. 95-1134 and RFBR grant No. 96-0449439. References 1 Witman, G.B. (1993) Chlamydomonas phototaxis, Trends Cell Biol. 3, 403–408 2 Kreimer, G. (1994) Cell biology of phototaxis in flagellate alga, Int. Rev. Cytol. 148, 229–310 3 Hegemann, P. (1997) Vision in microalgae, Planta 203, 265–274 4 Melkonian, M. and Robenek, H. (1984) The eyespot apparatus of flagellate green algae: a critical review, Prog. Phycol. Res. 3, 193–268 5 Foster, K-W. and Smyth, R.D. (1980) Light antennas in phototactic algae, Microbiol. Rev. 44, 572–630 6 Morel-Laurens, N.M.L. and Feinleib, M.E. (1983) Photomovement in an ‘eyeless’ mutant of Chlamydomonas, Photochem. Photobiol. 37, 189–194 7 Kreimer, G. and Melkonian, M. (1990) Reflection confocal laser scanning microscopy of eyespots in flagellated green algae, Eur. J. Cell Biol. 53, 101–111 8 Schaller, K. and Uhl, R. (1997) A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas, Biophys. J. 73, 1573–1578
trends in plant science reviews 9 Sineshchekov, O.A. (1991) Electrophysiology of photomovements in flagellated algae, in Biophysics of Photoreceptors and Photomovements in Microorganisms (Lenci, F. et al., eds), pp. 191–202, Plenum Press, New York 10 Yoshimura, K. (1994) Chromophore orientation in the photoreceptor of Chlamydomonas as probed by stimulation with polarized light, Photochem. Photobiol. 60, 594–597 11 Sineshchekov, O.A. (1991) Photoreception in unicellular flagellates: bioelectric phenomena in phototaxis, in Light in Biology and Medicine (Vol. 2) (Douglas, R.H., ed.), pp. 523–532, Plenum Press, New York 12 Harz, H., Nonnengaesser, C. and Hegemann, P. (1992) The photoreceptor current in the green alga Chlamydomonas, Philos. Trans. R. Soc. London Ser. B 338, 39–52 13 Litvin, F.F., Sineshchekov, O.A. and Sineshchekov, V.A. (1978) Photoreceptor electric potential in the phototaxis of the alga Haematococcus pluvialis, Nature 271, 476–478 14 Sineshchekov, O.A. et al. (1992) Photoelectric responses in phototactic flagellated algae measured in cell suspension, J. Photochem. Photobiol. B Biol. 13, 119–134 15 Harz, H. and Hegemann, P. (1991) Rhodopsin-regulated calcium currents in Chlamydomonas, Nature 351, 489–491 16 Braun, F-J. and Hegemann, P. Two independent photoreceptor currents in the spheroid alga Volvox carteri, Biophys. J. (in press) 17 Rueffer, U. and Nultsch, W. (1991) Flagellar photoresponses of Chlamydomonas cells held on micropipettes: II. Change in flagellar beat pattern, Cell Motil. Cytoskeleton 18, 269–278 18 Sineshchekov, O.A., Litvin, F.F. and Keszthelyi, L. (1990) Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis, Biophys. J. 57, 33–39 19 Sineshchekov, O.A. et al. (1994) Photoinduced electric currents in carotenoiddeficient Chlamydomonas mutants reconstituted with retinal and its analogs, Biophys. J. 66, 2073–2084 20 Holland, E-M. et al. (1996) The nature of rhodopsin-triggered photocurrents in Chlamydomonas. I. Kinetics and influence of divalent cations, Biophys. J. 70, 924–931 21 Schlicher, U. et al. (1995) G proteins and Ca21-modulated protein kinases of a plasma membrane-enriched fraction and isolated eyespot apparatuses of Spermatozopsis similis (Chlorophyceae), Eur. J. Phycol. 30, 319–330 22 Calenberg, M. et al. (1998) Light- and Ca21-modulated heteromeric GTPases in the eyespot apparatus of a flagellate green alga, Plant Cell 10, 91–103 23 Linden, L. and Kreimer, G. (1995) Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis, Planta 197, 343–351 24 Horst, C.J. and Witman, G.B. (1993) Ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance, J. Cell Biol. 120, 733–741 25 Rueffer, U. and Nultsch, W. (1997) Flagellar photoresponses of ptx1, a nonphototactic mutant of Chlamydomonas, Cell Motil. Cytoskeleton 37, 111–119
26 Pazour, G.J., Sineshchekov, O.A. and Witman, G.B. (1995) Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii, J. Cell Biol. 131, 427–440 27 King, S.J. and Dutcher, S.K. (1997) Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains, J. Cell Biol. 136, 177–191 28 Beck, C. and Uhl, R. (1994) On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii, Biophys. J. 125, 1119–1125 29 Nonnengaesser, C. et al. (1996) The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent cations, Biophys. J. 70, 932–938 30 Matsuda, A. et al. (1998) Isolation and characterization of novel mutants of Chlamydomonas that display phototaxis but not photophobic response, Cell Motil. Cytoskeleton 41, 353–362 31 Holland, E-M. et al. (1997) Control of phobic behavioural responses by rhodopsin-induced photocurrents in Chlamydomonas, Biophys. J. 73, 1359–1401 32 Yoshimura, K., Shingyoji, C. and Takahashi, K. (1997) Conversion of beating mode in Chlamydomonas flagella induced by electric stimulation, Cell Motil. Cytoskeleton 36, 236–245 33 Govorunova, E.G., Sineshchekov, O.A. and Hegemann, P. (1997) Desensitization and dark recovery of the photoreceptor current in Chlamydomonas reinhardtii, Plant Physiol. 115, 633–642 34 Zacks, D.N. et al. (1993) Comparative study of phototactic and photophobic receptor chromophore properties in Chlamydomonas reinhardtii, Biophys. J. 65, 508–518 35 Zacks, D.N. and Spudich, J.L. (1994) Gain setting in Chlamydomonas reinardtii: mechanism of phototaxis and the role of the photophobic response, Cell Motil. Cytoskeleton 29, 225–230 36 Foster, K-W. et al. (1984) A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas, Nature 311, 756–759 37 Spudich, J.L., Zacks, D. and Bogomolni, R. (1995) Microbial sensory rhodopsins: photochemistry and function, Isr. J. Chem. 35, 495–513 38 Deininger, W. et al. (1995) Chlamyrhodopsin represents a new type of sensory photoreceptor, EMBO J. 23, 5849–5858 39 Takahashi, T. and Watanabe, M. (1993) Photosynthesis modulates the sign of phototaxis of wild-type Chlamydomonas reinhardtii, FEBS Lett. 336, 516–520 40 Sineshchekov, O.A. and Govorunova, E.G. (1991) Rhythmic motion activity of unicellular flagellated algae and its role in phototaxis, Biophizika 36, 603–608
Oleg A. Sineshchekov* and Elena G. Govorunova are at the Biology Faculty, Moscow State University, 119899 Moscow, Russia.
*Author for correspondence (tel 17 095 9395489; e-mail [email protected]).
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45 Press, C.M. et al. (1997) Salicylic acid produced by Serratia marcescens 90-166 is not the primary determinant of induced systemic resistance in cucumber or tobacco, Mol. Plant–Microbe Interact. 10, 761–768 46 Pieterse, C.M.J. et al. (1998) A novel signaling pathway controlling induced systemic resistance in Arabidopsis, Plant Cell 10, 1571–1580 47 Ryals, J.A. et al. (1997) The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor IkB, Plant Cell 9, 425–439 48 Cao, H. et al. (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats, Cell 88, 57–63 49 Knoester, M. et al. (1998) Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi, Proc. Natl. Acad. Sci. U. S. A. 95, 1933–1937 50 McConn, M. et al. (1997) Jasmonate is essential for insect defense in Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 94, 5473–5477 51 Staswick, P.E., Yuen, G.Y. and Lehman, C.C. (1998) Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare, Plant J. 15, 747–745 52 Vijayan, P. et al. (1998) A role for jasmonate in pathogen defense of Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 95, 7209–7214 53 Thomma, B.P.H.J. et al. (1998) Separate jasmonate-dependent and salicylatedependent defense response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens, Proc. Natl. Acad. Sci. U. S. A. 95, 15107–15111
Corné M.J. Pieterse* and Leendert C. van Loon are at the Section of Plant Pathology, Dept of Plant Ecology and Evolutionary Biology, Utrecht University, PO Box 800.84, 3508 TB Utrecht, The Netherlands.
*Author for correspondence (tel 131 30 253 6887; fax 131 30 251 8366; e-mail [email protected]; internet http://www.bio.uu.nl/~fytopath).
Rhodopsin-mediated photosensing in green flagellated algae Oleg A. Sineshchekov and Elena G. Govorunova Green flagellated algae possess a primitive visual system that regulates the activity of their motor apparatus. Photoexcitation of a rhodopsin-type photoreceptor protein gives rise to the photoreceptor current, which, above a certain threshold of stimulus intensity, induces the flagellar current. It is probable that the photoinduced alteration in flagellar beating is governed by changes in intracellular Ca21 concentration. This rhodopsin-mediated sensory system serves to align the swimming path with the direction of the light stimulus, whereas processes of energy metabolism determine whether the oriented movement is directed towards or away from the light source.
S
urvival of phototrophic organisms depends on their ability to optimize their exposure to sunlight. Light controlled cell motility appeared early in evolution as one of the strategies used to achieve this goal. Unicellular flagellated algae, as well as motile zoospores and gametes of macroalgae, actively search for optimal light conditions by means of phototaxis (i.e. oriented swimming towards or away from the light source)1,2. In addition, a sudden change in light intensity, irrespective of its direction, usually elicits a photophobic or photoshock cell response, which
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appears as a transient stop, often followed by a brief period of backward motion1. Most flagellates rotate around their longitudinal axis during forward swimming. The ability to track the direction of light is based on the capacity to sense a temporal difference in the amount of quanta captured in a single photoreceptive region of the cell under lateral illumination. Structural and functional aspects of phototactic sensory systems found in different taxonomic groups of algae are very diverse2 and might reflect their independent evolutionary origin. Chlorophyceae are a unique group of
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(98)01370-3
trends in plant science reviews microorganisms, because the photosensory transduction mechanisms found in these algae are strikingly similar to the processes of animal vision2,3. Therefore, elucidation of these mechanisms might give a new insight into the evolution of photosensory systems. Photoreceptor apparatus
Light
Flagella
?
Unbalanced flagellar response (phototaxis)
Eyespot PC 2 Green flagellated algae usually carry an 221111 2 FC Rhodopsin PC 12 12 eyespot, seen under the light microscope as 12 photoexcitation generation 2 1111 2 a highly pigmented organelle from about 22 2 2 one to a few mm in size. The eyespot is Electrotonic often located at the cell equator within the spread of membrane chloroplast close to the cell surface, and is depolarization FC Flagellar displaced by 20–408 from the plane of flageneration undulation gellar beating. The precise position of the (photophobic eyespot is probably ensured by its strucresponse) tural association with the flagellar root sysFig. 1. Rhodopsin-mediated signal transduction chains for phototaxis and photophobic tem4. The eyespot consists of one to several response in green flagellated algae. Photoexcitation of the photoreceptor rhodopsin leads to layers of carotenoid globules, often subgeneration of the photoreceptor current (PC) across the eyespot region of the cell memtended by specialized thylakoid membrane. PC generation is responsible for phototaxis, which appears to be based on differen4,5 branes . The visible pigment is, however, tial sensitivity of the axonemes to Ca21. The molecular mechanism of the coupling between not the photoreceptor pigment, which is the PC generation and the unbalanced flagellar response has yet to be established. When present in a much lower concentration and light stimulus intensity exceeds the critical level necessary to evoke the photophobic response, a flagellar current (FC) is observed. Ca21 channels in the flagellar membrane are is assumed to be embedded in the plasma activated by the PC-induced depolarization, which electrotonically spreads over the whole membrane and/or the outer chloroplast enplasma membrane. Changes in the membrane potential, induced by generation of the PC velope membrane in the area overlaying 4 and the FC, can so far only be indirectly estimated. Therefore the distribution of the positive the eyespot . The photoreceptor membranes and negative charges shown here corresponds to the direction of the change (i.e. the resting and the eyespot together form the photorepotential becomes less negative), and not to the absolute value of the potential upon photoceptor apparatus. The importance of the excitation. The FC is carried by a massive influx of Ca21 ions, which leads to conversion of eyespot for normal phototactic orientation flagellar beating mode from ‘breast stroke’ to undulation. has been demonstrated in experiments with an eyespot-deficient Chlamydomonas mutant6. When the photoreceptor apparatus is turned away from the light source, photoreceptor illumination is and a ‘dissolver’ Volvox mutant16, whereas the suspension method attenuated by absorption and scattering of light by the eyespot. In of photoelectric measurements is more universal. Common feaaddition, multi-layered eyespots, present in many species of green tures of the photoelectric responses found in different species sugflagellates, appear to be quarter-wave interference reflectors, gest that the same basic scheme of sensory transduction holds for which produce maximum light intensity at the presumed photo- the whole group of chlorophyceae (Fig. 1). receptor site when the photoreceptor apparatus is facing the light The earliest detectable event in the signal transduction chain stimulus5,7. Illumination of the photoreceptor does not change dur- for both phototaxis and photophobic response is the generation of ing the rotation cycle when the cell swims parallel to the light di- an inward current across the portion of the plasma membrane rection, but becomes periodic when the cell deviates from it. This overlaying the eyespot, defined as the photoreceptor current (PC; periodic signal is perceived as a stimulus for correcting the swim- Refs 13,15). Switching on the light induces a transient PC peak ming path5. Light absorption by the bulk chloroplast pigments also which decays to a lower stationary level and dissipates after contributes to modulation of the photoreceptor illumination6,8. switching off the light (Fig. 2). The action spectrum for PC genFinally, preferential absorption (dichroism) of the photoreceptor eration is very close to the action spectra for photomotile cell itself, resulting from the highly ordered orientation of the chromo- responses. The role of the PC in phototaxis was revealed by phores9,10, probably adds to the directivity of the photoreceptor correlation of electrical responses to photoinduced changes in apparatus5. flagellar beating observed in a cell held on a pipette. Step-up stimulus induced an increase in the beat frequency of the cisSignal transduction mechanisms flagellum (the one closest to the eyespot), and a decrease in that of Electrical processes play a key role in photosensory transduction the trans-flagellum, whereas step-off stimulus caused the oppoin green flagellated algae9,11,12. Algal cells do not usually exceed site effect9,11. Photoinduced changes in front amplitude, which 10–20 mm in diameter, which makes microelectrode recording were opposite in the cell’s two flagella, were also reported9,11,17. rather problematic. Instead, the photoinduced electrical signals Such unbalanced motor responses of the two flagella would lead involved in phototaxis and photophobic response in green flagel- to the correction of the swimming path in a freely swimming cell lates can be measured extracellularly from individual cells13 and (i.e. to phototaxis). from cell suspensions14. Extracellular recording takes advantage Complex kinetics of the PC (Ref. 18) and the biphasic deof the asymmetric localization of the signal sources within the pendence of its peak amplitude on stimulus intensity11,14,19 led to cell. Application of the suction pipette technique is limited to rela- the conclusion that two different electrical processes contribute tively large flagellates with elastic cell walls such as wild-type to PC generation9,11. Upon flash excitation, the first process deHaematococcus13, cell wall-deficient Chlamydomonas mutants15 velops within the time resolution of the measuring system (,5 ms; February 1999, Vol. 4, No. 2
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Light off
(a)
Current (nA)
Light on
15 1.8
0.2
0.0
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FC
Current (pA)
10 PC 5
0.00 0.02 Time (m s21)
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0
3 0
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2
25
20
30
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500 Time (m
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Fig. 2. Photoelectric responses elicited in the same cell of Haematococcus pluvialis by a pulse of continuous light at two different fluence rates:1, 7.5 3 1019 photons m22 s21; 2, 1.75 3 1019 photons m22 s21. FC; flagellar current: PC; photoreceptor current.
Fig. 3). Its peak amplitude saturates at extremely high light intensities, indicating that it is limited only by photoconversion of the photoreceptor protein11. The delay time of the second process increases with the decrease in flash intensity and could extend to a few ms (Fig. 3), indicating the involvement of intermediate biochemical steps in signal transduction. Light saturation of its peak amplitude can be up to three orders of magnitude lower than that of the first process, and its maximum peak amplitude reaches only about 10% of that of the first process (Fig. 3). The two processes that contribute to PC generation were defined, according to their features, as the ‘early PC’ and the ‘late PC’ (or, alternatively, as the ‘high-saturating PC’ and the ‘low-saturating PC’). At high stimulus intensity, the recorded PC mostly consists of the early PC, whereas at low stimulus intensity, the late PC makes a major contribution to the recorded signal. Especially long delay times for PC at low stimulus intensity have been recently reported in Volvox16. At high stimulus intensity, the number of elementary charges transported across the photoreceptor membrane (calculated from the area under the curve of PC) is close to the number of photons absorbed by the photoreceptor. Taking the virtually instant onset of the early PC into account, there are two possibilities, either the early PC results from translocation of the ions across the membrane by a low-conductance ion channel closely associated with the rhodopsin12, or even by the rhodopsin itself. At physiological conditions PC is mostly driven by the influx of Ca21 ions13,15, although a Ca21-independent component is also present13,20, which in Volvox has been defined as a ‘slow PC’ (Ref. 16). A drop in the external pH dramatically increases PC even in the absence of external Ca21, which points to the contribution of H1 influx to PC generation, rather than to acid facilitated Ca21 influx16. The amplitude of the stationary current, which is probably determined by the late PC, corresponds to about 107 elementary charges per s being transported across the membrane when 103 quanta per s are absorbed by the photoreceptor9,11. This means that generation 60
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High-saturating early PC
4 Peak current (nA)
0
2
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0 0.001
0.01
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1
10
100
Flash fluence (relative units)
Fig. 3. (a) Photoelectric responses evoked in a cell suspension of Chlamydomonas reinhardtii strain 495 (1) at different flash intensities. Photo-flash excitation, relative stimulus intensity: 1, 100%; 2, 2.5%; 3, 0.01%. Arrows indicate the time of the flash. Broken line shows the response delay in trace 3. Inset: the onset of the photoreceptor current (PC) recorded with an improved time resolution in Chlamydomonas reinhardtii strain 516/white-3 supplemented with 1028 M all-trans retinal. Laser flash excitation, 500 nm, 2 J m22. The laser artifact is digitally subtracted. (b) Dependence of the PC peak amplitude measured in a cell suspension of Chlamydomonas reinhardtii strain 495 (1) on the fluence of the excitation flash. Solid line shows a computer fit to Michaelis function for the late PC and to exponential function for the early PC.
of the late PC involves up to four orders of signal amplification. The basis of the amplification cascade involved in generating the late PC is not yet known, but recent biochemical and immunological studies on isolated photoreceptor apparatuses point to the presence of G proteins, which are also involved in the enzymatic cascade of animal photosensory transduction21. Most convincingly, light-dependent GTPase activity, with an action spectrum similar to that of rhodopsin absorption, was found in isolated eyespot apparatuses of the green alga Spermatozopsis, which was inhibited by antibodies raised against Chlamydomonas rhodopsin22. In addition, Ca21-dependent protein kinase and phosphatase activities were found in a similar in vitro preparation21,23. Experiments in reactivated, demembranated Chlamydomonas cell models revealed different sensitivities of cis- and trans-axonemes
trends in plant science reviews
Direction
Sensory transduction
Modulation of photoreceptor illumination
Unbalanced flagellar response
Degree of orientation Cell alignment
Light
Cell orientation Movement along the intensity gradient
Intensity gradient
to Ca21 concentration, which might account for the unbalanced motor response of the two flagella, which is necessary for phototaxis1. This Ca21-dependent shift in flagellar dominance was not found in the cell models of a non-phototactic ptx1 mutant that lacks a pair of 75-kDa axonemal proteins24. In this mutant, photoinduced changes in beat frequency and amplitude occur in both flagella as in the trans-flagellum in the wild type25. The ptx5, ptx6 and ptx7 mutants probably have other defects in genes encoding axonemal proteins responsible for this shift26. Asymmetric phosphorylation of a 138-kDa protein of an inner dynein arm complex appears to be one more factor to determine different sensitivity of the two axonemes to Ca21 associated with phototaxis27. When the intensity of the light stimulus exceeds a certain threshold, PC becomes superimposed by an ‘all-or-nothing’ spikelike response (Fig. 2). The increase in stimulus intensity shortens its delay time from over 60 down to 5 ms (Refs 13,15). It reflects an inward current across the flagellar membrane and is therefore defined as the ‘flagellar current’ (FC; Ref. 15). No FC could be recorded immediately after complete mechanical amputation of flagella, and the time course of the signal recovery was found to correlate with that of flagellar regrowth28. The area under the curve of PC before the beginning of the FC is nearly constant over a wide range of stimulus intensities9,11,12. This indicates that a certain amount of charge should enter the cell to initiate the FC. When the cell is hyperpolarized by photosynthetically-active red background illumination, this integral increases twofold9,11. Therefore it could be concluded that FC is activated by the PC-induced depolarization of the membrane to a certain level. FC is more sensitive to the removal of Ca21 from external medium than PC (Ref. 13), and is most probably driven by the activation of voltage-gated Ca21-channels in the flagellar membrane15,28. FC can also be observed at low external pH in Ca21-free medium, which indicates that flagellar channels are partially permeable to protons29. They are likely to be perturbed in ppr1–ppr4 mutations in Chlamydomonas30, whereas ptx2 and ptx8 strains probably lack an element in the signal transduction chain, which is located downstream from PC generation and is common for the phototaxis and the photophobic response pathways26. FC drives a switch from ‘breast-stroke’ flagellar beating to flagellar undulation, which is characteristic of the photophobic response in a freely swimming cell. This notion has been proven by simultaneous recording of photocurrents and flagellar beating from the same cell held on a micropipette31. No spike-like FC was found in Volvox, the cells of which react to photophobic stimuli only by a transient arrest of flagellar beating rather than by a switch to flagellar undulation16. Externally applied electrical depolarization activates flagellar Ca21 channels and induces conversion of beating mode in Chlamydomonas flagella32. Spontaneous spikes similar to FC can sometimes be observed in the dark or under continuous illumination13,31, which might play a role in regulation of spontaneous directional changes (klinokinesis). Studies on isolated, reactivated flagellar apparatuses and axonemes have established that Ca21 acts directly on the axoneme and at around 1024 M induces a switch from ciliary type stroke to undulation, probably involving Ca21-dependent phosphorylation of specific axonemal proteins1–3. The spike-like FC, also called the ‘fast flagellar current’ (Ff), is accompanied by the ‘slow flagellar current’ (Fs) with a peak time of a few hundred ms12, the amplitude of which dramatically increases upon substitution of external Ca21 for Ba21 (Ref. 20). The functional role of Fs is presumably related to controlling the duration of backward swimming during the photophobic response12,20.
Control of the response sign
Sign of phototaxis Cell illumination level
Energetic state of cell
Photosynthesis
Fig. 4. Two feedback loops of light controlled behaviour in green flagellated algae. Upper circle: a rhodopsin-mediated sensory system is responsible for the alignment of the swimming path with respect to the light stimulus. Tracking the light direction is based on modulation of the photoreceptor illumination during the rotation cycle. Deviation of the swimming path from the stimulus direction induces a change in the pattern of photoreceptor illumination, which initiates a corrective motile response. Lower circle: absorption of the light by photosynthetic apparatus determines the energy level of the cell, which controls the resting membrane potential. The resting membrane potential is involved in regulation of the sign of phototaxis (swimming towards or away from the light source). The net behavioural response results from a combination of the rhodopsin-based and photosynthesis-driven regulatory processes and leads to an accumulation of the cells in the area of optimal illumination.
Generation of Fs and stationary PC seems to be linked to K1 efflux, which counterbalances depolarization of the membrane caused by these long-lasting inward currents16,29. Transient K1 currents are triggered by membrane depolarization induced by Ff (Ref. 33); their direction can be inward or outward depending on the K1 electrochemical driving force. Photosensory adaptation
The phototaxis response does not become desensitized upon repetitive light stimulation, which leads to prominent desensitization of the photophobic response34. Precise desensitization of the photophobic response at high stimulus intensities provides the large dynamic range of phototaxis35. Desensitization and dark recovery of the cell upon photoexcitation is determined by changes in the membrane potential33. FC, which is only necessary for the photophobic response, causes stronger depolarization of the plasma membrane than PC alone, and consequently increases the refractory period upon light stimulation. One of the recovery mechanisms involved in restoration of the resting membrane potential after depolarization induced by FC, is an outward K1 current observed at low external K1 concentrations33. February 1999, Vol. 4, No. 2
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trends in plant science reviews Photoreceptor protein
Initial information on the nature of the photoreceptor responsible for phototaxis in green flagellated algae has been obtained by action spectroscopy. The maximum efficiency of wavelengths of around 500 nm for most green flagellates favoured the view that carotinoproteins were the photoreceptor pigments. The hypothesis has been suggested that these proteins might resemble rhodopsins involved in animal vision5. This has been confirmed by experiments in which phototaxis in blind carotenoid-deficient Chlamydomonas mutants was restored upon the addition of exogenous retinal and its analogues36. Reconstitution studies undertaken with a variety of retinoid compounds, discussed in detail in a recent review37, led to the conclusion that all-trans, 6-s-trans retinal is the native chromophore in the Chlamydomonas rhodopsin as in archaebacterial rhodopsins. Photoexcitation gives rise to its isomerization to a 13-cis form, which again resembles archaebacterial rhodopsins, whereas the primary event in animal vision is isomerization of 11-cis-retinal to all-trans-retinal. The functional Chlamydomonas photoreceptor requires the presence of at least three conjugated double bonds and the 13-methyl group in the polyene chain of its chromophore. The addition of all-transretinal, 9-dimethyl-retinal and dimethyl-octatrienal results in the appearance of the normal PC, which proves that restoration of behavioural responses is indeed due to reconstitution of the functional photoreceptor19. Hydroxylamine, the agent known to induce light-dependent cleavage of the chromophore in retinal-containing proteins, causes a specific inhibition of phototaxis and PC in wildtype cells3,19. These in vivo results have been complemented by HPLC detection of retinoids in cell extracts2,3. A single 30-kDa protein was identified in Chlamydomonas carotenoid-deficient cells by 3Hretinal labelling38. Application of a similar procedure to the eyespot fraction isolated from wild-type cells allowed purification of the presumed photoreceptor protein to electrophoretic homogeneity38. Polyclonal antibodies raised against Chlamydomonas opsin concentrated precisely in the eyespot area in permeabilized cells thus indicating localization of the rhodopsin in vivo38. The opsin cDNA was purified and sequenced38, and genomic opsin clones were isolated from Chlamydomonas and Volvox3. The derived opsin amino acid sequences are 65% identical and show no homology to bacterial opsins, in spite of the similarity in chromophore properties described above. On the other hand, some homology to animal opsins, especially those from invertebrates, was apparent. Algal opsin sequences are enriched in polar amino acid residues and bear some homology with ion channel sequences38, which corroborates the above hypothesis that the rhodopsin itself might form the light-regulated membrane conductance. Regulation of phototaxis by photosynthesis and other processes of energy metabolism
Phototaxis in green flagellated algae is mediated by a specific photoreceptor system that is separate from the photosynthetic apparatus. However, cell accumulation in the area of optimal light conditions for phototrophic metabolism requires the involvement of a negative feedback loop in light control of photomovement. This loop is provided by regulation of phototaxis by photosynthesis and other energy conversion processes (Fig. 4). A rapid switch from positive to negative phototaxis (swimming towards, or away from the light source, respectively) can be induced by a red background illumination that is by itself insufficient for photoorientation19,39. Red light induces hyperpolarization of the plasma membrane and the increase in PC amplitude18, which leads to the reversal of phototaxis sign. Spontaneous changes of swimming direction about every 25–30 s were observed both in dark and at 62
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moderate stimulus intensities in Chlamydomonas and Haematococcus40. This behaviour is sensitive to red light, and is probably related to the periodic electrical processes in the plasma membrane detected by extracellular microelectrodes9. A phase shift between the two periodic electrical processes, presumably associated with the two flagella of the cell observed upon turning on the light might control the switch from positive to negative phototaxis. Prospects
Although a general overview of the signalling pathways involved in light control of behaviour in green flagellated algae has been outlined in recent years, identification and characterization of individual molecular components of the photosensory system has only just begun. Detailed genetic analysis and phenotypic characterization of mutants defective in phototaxis and photophobic response should help to dissect the rhodopsin-mediated signal transduction chain. Sequencing opsins from Chlamydomonas and Volvox provides the basis for genetic engineering of the key elements of this chain. Overexpression of opsins and other gene products involved in photosensory transduction in green flagellates is probably the only way in which to collect enough protein for spectroscopic and biochemical studies. The physiological significance of individual components of the signalling pathway should be verified by investigating photosensory function in vivo by means of non-invasive physiological techniques such as photoelectric measurements in cell suspensions. Physiological studies on intact cells would also help to elucidate how the photosensory system is integrated in general cell metabolism. By extending the research to a wider range of species it might be possible to identify subjects that are more amenable for comparative analysis and suitable for modern experimental techniques, such as patch clamping and the use of ion-specific fluorescent indicators. Comparative analysis of signal transduction mechanisms in different species of green flagellates is highly desirable for understanding the evolution of rhodopsin-mediated signalling systems. Acknowledgements
We would like to thank Dr F-J. Braun and Prof. P. Hegemann for providing us with their manuscript submitted for publication, and Dr G. Kreimer, Dr U. Rueffer and Prof. W. Nultsch for the reprints of their recent work. We thank I.M. Altschuler for his help in preparation of the manuscript. This work was supported by INTAS-RFBR grant No. 95-1134 and RFBR grant No. 96-0449439. References 1 Witman, G.B. (1993) Chlamydomonas phototaxis, Trends Cell Biol. 3, 403–408 2 Kreimer, G. (1994) Cell biology of phototaxis in flagellate alga, Int. Rev. Cytol. 148, 229–310 3 Hegemann, P. (1997) Vision in microalgae, Planta 203, 265–274 4 Melkonian, M. and Robenek, H. (1984) The eyespot apparatus of flagellate green algae: a critical review, Prog. Phycol. Res. 3, 193–268 5 Foster, K-W. and Smyth, R.D. (1980) Light antennas in phototactic algae, Microbiol. Rev. 44, 572–630 6 Morel-Laurens, N.M.L. and Feinleib, M.E. (1983) Photomovement in an ‘eyeless’ mutant of Chlamydomonas, Photochem. Photobiol. 37, 189–194 7 Kreimer, G. and Melkonian, M. (1990) Reflection confocal laser scanning microscopy of eyespots in flagellated green algae, Eur. J. Cell Biol. 53, 101–111 8 Schaller, K. and Uhl, R. (1997) A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas, Biophys. J. 73, 1573–1578
trends in plant science reviews 9 Sineshchekov, O.A. (1991) Electrophysiology of photomovements in flagellated algae, in Biophysics of Photoreceptors and Photomovements in Microorganisms (Lenci, F. et al., eds), pp. 191–202, Plenum Press, New York 10 Yoshimura, K. (1994) Chromophore orientation in the photoreceptor of Chlamydomonas as probed by stimulation with polarized light, Photochem. Photobiol. 60, 594–597 11 Sineshchekov, O.A. (1991) Photoreception in unicellular flagellates: bioelectric phenomena in phototaxis, in Light in Biology and Medicine (Vol. 2) (Douglas, R.H., ed.), pp. 523–532, Plenum Press, New York 12 Harz, H., Nonnengaesser, C. and Hegemann, P. (1992) The photoreceptor current in the green alga Chlamydomonas, Philos. Trans. R. Soc. London Ser. B 338, 39–52 13 Litvin, F.F., Sineshchekov, O.A. and Sineshchekov, V.A. (1978) Photoreceptor electric potential in the phototaxis of the alga Haematococcus pluvialis, Nature 271, 476–478 14 Sineshchekov, O.A. et al. (1992) Photoelectric responses in phototactic flagellated algae measured in cell suspension, J. Photochem. Photobiol. B Biol. 13, 119–134 15 Harz, H. and Hegemann, P. (1991) Rhodopsin-regulated calcium currents in Chlamydomonas, Nature 351, 489–491 16 Braun, F-J. and Hegemann, P. Two independent photoreceptor currents in the spheroid alga Volvox carteri, Biophys. J. (in press) 17 Rueffer, U. and Nultsch, W. (1991) Flagellar photoresponses of Chlamydomonas cells held on micropipettes: II. Change in flagellar beat pattern, Cell Motil. Cytoskeleton 18, 269–278 18 Sineshchekov, O.A., Litvin, F.F. and Keszthelyi, L. (1990) Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis, Biophys. J. 57, 33–39 19 Sineshchekov, O.A. et al. (1994) Photoinduced electric currents in carotenoiddeficient Chlamydomonas mutants reconstituted with retinal and its analogs, Biophys. J. 66, 2073–2084 20 Holland, E-M. et al. (1996) The nature of rhodopsin-triggered photocurrents in Chlamydomonas. I. Kinetics and influence of divalent cations, Biophys. J. 70, 924–931 21 Schlicher, U. et al. (1995) G proteins and Ca21-modulated protein kinases of a plasma membrane-enriched fraction and isolated eyespot apparatuses of Spermatozopsis similis (Chlorophyceae), Eur. J. Phycol. 30, 319–330 22 Calenberg, M. et al. (1998) Light- and Ca21-modulated heteromeric GTPases in the eyespot apparatus of a flagellate green alga, Plant Cell 10, 91–103 23 Linden, L. and Kreimer, G. (1995) Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis, Planta 197, 343–351 24 Horst, C.J. and Witman, G.B. (1993) Ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance, J. Cell Biol. 120, 733–741 25 Rueffer, U. and Nultsch, W. (1997) Flagellar photoresponses of ptx1, a nonphototactic mutant of Chlamydomonas, Cell Motil. Cytoskeleton 37, 111–119
26 Pazour, G.J., Sineshchekov, O.A. and Witman, G.B. (1995) Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii, J. Cell Biol. 131, 427–440 27 King, S.J. and Dutcher, S.K. (1997) Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains, J. Cell Biol. 136, 177–191 28 Beck, C. and Uhl, R. (1994) On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii, Biophys. J. 125, 1119–1125 29 Nonnengaesser, C. et al. (1996) The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent cations, Biophys. J. 70, 932–938 30 Matsuda, A. et al. (1998) Isolation and characterization of novel mutants of Chlamydomonas that display phototaxis but not photophobic response, Cell Motil. Cytoskeleton 41, 353–362 31 Holland, E-M. et al. (1997) Control of phobic behavioural responses by rhodopsin-induced photocurrents in Chlamydomonas, Biophys. J. 73, 1359–1401 32 Yoshimura, K., Shingyoji, C. and Takahashi, K. (1997) Conversion of beating mode in Chlamydomonas flagella induced by electric stimulation, Cell Motil. Cytoskeleton 36, 236–245 33 Govorunova, E.G., Sineshchekov, O.A. and Hegemann, P. (1997) Desensitization and dark recovery of the photoreceptor current in Chlamydomonas reinhardtii, Plant Physiol. 115, 633–642 34 Zacks, D.N. et al. (1993) Comparative study of phototactic and photophobic receptor chromophore properties in Chlamydomonas reinhardtii, Biophys. J. 65, 508–518 35 Zacks, D.N. and Spudich, J.L. (1994) Gain setting in Chlamydomonas reinardtii: mechanism of phototaxis and the role of the photophobic response, Cell Motil. Cytoskeleton 29, 225–230 36 Foster, K-W. et al. (1984) A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas, Nature 311, 756–759 37 Spudich, J.L., Zacks, D. and Bogomolni, R. (1995) Microbial sensory rhodopsins: photochemistry and function, Isr. J. Chem. 35, 495–513 38 Deininger, W. et al. (1995) Chlamyrhodopsin represents a new type of sensory photoreceptor, EMBO J. 23, 5849–5858 39 Takahashi, T. and Watanabe, M. (1993) Photosynthesis modulates the sign of phototaxis of wild-type Chlamydomonas reinhardtii, FEBS Lett. 336, 516–520 40 Sineshchekov, O.A. and Govorunova, E.G. (1991) Rhythmic motion activity of unicellular flagellated algae and its role in phototaxis, Biophizika 36, 603–608
Oleg A. Sineshchekov* and Elena G. Govorunova are at the Biology Faculty, Moscow State University, 119899 Moscow, Russia.
*Author for correspondence (tel 17 095 9395489; e-mail [email protected]).
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