PIanta (Berl.) 111, 167--178 (1973) 9 by Springer-Verlag 1973
Phototaxis in a Dinoflagellate: Action Spectra as Evidence for a Two-Pigment System* R i c h a r d B. F o r w a r d , Jr. Zoology Department, Duke University Marine Laboratory, Beaufort, North Carolina 28516, U.S.A. Received October 5, 1972 / January 31, 1973
Summary. Action spectra were determined in the UV region of the spectrum for the first phase of the phototactie response (stop response) and for the phytochrome pigment associated with this response in the dinoflageltate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after F R irradiation they shift to 490 and 300-310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of PI~ in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The PFR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of PI~ to PFR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.
Introduction The dinoflagellate Gyrodinium dorsum K o f o i d r e s p o n d s in a p h o t o t a c t i c m a n n e r t o light s t i m u l a t i o n . A t high light intensities t h e response sequence consists of a n initial cessation of m o v e m e n t (stop response) followed b y s w i m m i n g in t h e d i r e c t i o n of t h e s t i m u l u s b e a m ( H a n d et al., !967). A similar, i n i t i a l cessation of m o v e m e n t u p o n s t i m u l a t i o n with l i g h t occurs in o t h e r p h y t o f l a g e l l a t e s , e. g., Chlamydomonas (Feinlieb a n d Curry, 1971) a n d Volvox ( I t u t h , 1970). I n all cases this s t o p response is considered a n i n t e g r a l p a r t of t h e p h o t o t a e t i c response, a n d t h u s i t can be used as a n i n d i c a t o r of p h o t o t a c t i c light p e r c e p t i o n . I n t h e visible region Gyrodinium is m o s t responsive to blue light; however, t h e exac~ m a x i m u m d e p e n d s u p o n w h e t h e r t h e cells are pre-
* Abbreviations. F R ~far-red, R ~ red, PFl~=far-red-absorbing form of phytochrome, P~=red-absorbing form of phytochrome, UV=ultraviolet.
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R . B . Forward, Jr. :
i r r a d i a t e d with R (620-nm) or F R (700-nm) light, a n d these effects of long-wave light are m u t u a l l y reversible ( F o r w a r d , 1970). This d e p e n d e n c e u p o n R a n d F R light a n d o t h e r evidence ( F o r w a r d a n d D a v e n p o r t , 1968) i n d i c a t e t h a t a p h y t o c h r o m e p i g m e n t is i n v o l v e d as a n a d d i t i o n a l f a c t o r in p h o t o t a x i s . A t e n t a t i v e m o d e l for t h e p h o t o t a e t i e p i g m e n t s y s t e m p r o p o s e d t h e i n t e r a c t i o n of two pigments, a n u n i d e n t i f i e d b l u e - a b s o r b i n g p i g m e n t for t h e directional response a n d a p h y t o c h r o m e controlling t h e s e n s i t i v i t y of t h e cells ( F o r w a r d , 1970). Since all p r e v i o u s studies w i t h Gyrodinium were c o n d u c t e d w i t h visible light, i t was t h o u g h t t h a t f u r t h e r i n f o r m a t i o n a b o u t t h e p i g m e n t s i n v o l v e d m i g h t be gained b y determining t h e a c t i o n s p e c t r a for b o t h t h e b l u e - a b s o r b i n g a n d t h e phy~ochrome p i g m e n t in the U V region. Such a n e x a m i n a t i o n is t h e s u b j e c t of the p r e s e n t investigation.
Methods and Materials The culture methods for Gyrodinium dorsum Kofoid remain as previously described (Hand et al., 1967). Cells were grown in a model CEL-44 growth chamber (Sherer Gillet, Marshall, Mich., U.S.A.) with a light-dark cycle of 16:8 h and at a temperature of 19~ Since responsiveness varies with culture age, only 6-d-old cultures were used, and all tests were begun 3 h after the onset of the light period. The equipment for monitoring photoresponses of individual cells resembles that described by Feinleib and Curry (1967). Cells are observed in dark field under a model UPL inverted microscope (Karl Zeiss, New York, N. Y.). The dark field illumination light is filtered with either a 621 or a 701-nm interference filter (Optics Technology, Palo Alto, Calif., U.S.A.). The light stimulus source is a model f/3.5 monochromator (Farrand Optical Co., Mount Vernon, N.Y., U.S.A.), coupled to a 150-W Xenon arc lamp. Except where noted, the full band pass for stimulus wavelengths is 10 nm. This light is focused onto the experimental vessel (florimeter cell made of infrasil quartz; interior dimensions 1 0 x 5 x 5 mm) by means of quartz lenses (Oriel Optics Corp., Stamford, Conn., U.S.A.), and its spectral purity regulated by Coming filters (Coming Glass Works, Coming, N. Y., U.S.A.), namely for the UV region, No. 7-54; for the blue, No. 4-96; and for the yellow-red region, No. 3-67. Since the arc position of the lamp varied with each experiment, intensities of the experimental wavelengths were measured before each experiment, using a model 65A radiometer (Yellow Springs Instrument Co., Yellow Springs, O., U.S.A.). Stimulus duration is controlled by a Uniblitz electromagnetic shutter (Vincent Associates, Rochester, N.Y., U.S.A.) which is set at 1.2 s. The intensity of the stimulus light is controlled by neutral density filters. In all experiments the initial stop response is used as the index of photoresponsiveness. Recording the responses was accomplished by photographing through the microscope with a 35-ram camera using Kodak-2475 recording film. Since the response latency is about 0.4 s, the solenoid for triggering the camera was activated 0.4 s after the beginning of light stimulation, as timed by an electronic timing circuit. By using a 0.5-s exposure, responding cells can be easily differentiated from swimming cells on the photographic negative, and the percent response calculated.
Pho~otaxis in a DinofiagelIate
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Since the microscope dark-field-illumination light can potentially alter cell responsiveness, this illumination only occurred while the ceils were actually being stimulated and responses photographed. At all other times the microscope illumination light was prevented from irradiating the cells by the appropriate placement of light shields. This light was filtered with a 601-nm interference filter for the determination of the stop response action spectrum after R irradiation, and with a 701-nm filter in all other experiments. The response criteria remain as previously established (Forward and Davenport, 1968). A response in which more than 50 % of the cells cease moving upon stimulation is designated as a "positive stop response". In all experiments the lowest stimulus light intensity (threshold) which would initiate a positive stop response was used to measure phototactie sensitivity under different conditions. Since the exact procedure of pre-illumination and stimulation varied with each experiment, they will be described in detail in the following section.
Results
1. Action Spectra/or the Stop Response Following R and F R Irradiation The visible action spectrum for the positive stop response depends upon whether the cells are pre-irradiated with 1%(620-nm) or F R (700-nm) light (Forward, 1970). These action spectra were redetermined to include wavelengths in the UV, utilizing the following procedures. To convert the p h y t o c h r o m e to P~a, aliquot cell samples were r e m o v e d from the culture chamber and irradiated for I min with 620-nm light (average intensity of 4.92 • 10 z5 hv cm -~ s-Z). The cells were then left in darkness for 1 min after which t h e y were stimulated with either an increasing or decreasing sequence of intensities at selected wavelengths in the visible and UV. The lowest intensity at which a positive stop response could be elicited was designated as the threshold intensity for t h a t wavelength. Since sensitivity is k n o w n to change over time in darkness, the order of stimulus intensity presentation (increasing or decreasing) was alternated from one trial to the next. However, the observed thresholds for each procedure were usually identical. A new cell sample was used for each determination at each wavelength. Fig. I a shows t h a t response m a x i m a occur at 470 and 280-nm. To convert the p h y t o c h r o m e to P~, cells were irradiated with 620-nm light (average intensity 5.5 • 10 z5 hv cm -2 s -z) for 1.5 rain, followed b y a 700-nm irradiation (full b a n d pass 20-nm; average intensity 6.45 • 10 z5 hv cm -2 s -z) for the same time. The cells were then left in darkness for 1 rain, a n d the threshold for a positive response at the different wavelengths determined as described above. Fig. 1 b shows t h a t the overall sensitivity to light is reduced b y F R light, and the m a x i m a are shifted to 490 and 300-310 nm. The UV m a x i m u m was placed at 300-310 n m
170
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because the lowest percentage of t o t a l l y u n r e s p o n s i v e cultures was f o u n d u p o n s t i m u l a t i o n with these wavelengths. I t should also be n o t e d t h a t after the R - F R irradiations no response to 280 n m was observed.
2. UV Action Spectrum/or Pi~ Responsiveness to 470 n m can be abolished b y a 4 m i n exposure to this wavelength. However, the response can be r e a c t i v a t e d b y s u b s e q u e n t
Phototaxis in a Dinoflagellate
171
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conversion of Pa to PFg by exposure to I~ light (Forward and Davenport, 1968). The level of the returned sensitivity to blue (470 nm) light varies with magnitude of phytochrome conversion from Pit to PFg and therefore can be used as a sensitive indicator of conversion (Forward, 1970). Thus in this experiment the UV action spectrum for P~ is determined by measuring the reactivation of responsiveness to 470 nm by selected UV wavelengths after a prolonged blue exposure. The procedure was to abolish responsiveness to blue (470 nm) by a 4-rain exposure to this light (average intensity 12.4 • 1015 hv cm -a s-l). This was followed by a 3-rain exposure to selected wavelengths between 410 and 310 nm, set at approximately equal quantal levels (e.g., 350 nm ----8.7 • 10 Is hv cm -~ s-X). Then after 1 rain in darkness the threshold for a positive stop response to 470 nm was determined. Controls demonstrated that following the initial 4-rain blue irradiation a n d 4 rain in darkness, t h e level of s t o p p i n g u p o n s t i m u l a t i o n was t h a t e x p e c t e d for a r a n d o m response (20-35 %). P r o l o n g e d exposure to 290 a n d 280 n m was n o t possible since t h e cells d i e d a l m o s t i m m e d i a t e l y . Fig. 2 shows t h a t t h e a p p a r e n t p e a k for P ~ a b s o r p t i o n in t h e n e a r U V is a t 320 nm, since g r e a t e s t s e n s i t i v i t y to 470 n m occurs a f t e r i r r a d i a t i o n
172
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with this wavelength. To completely illustrate the action spectrum for P~, the visible portion (from F o r w a r d and Davenport, 1968) is plotted on the right of Fig. 2.
3. UV Action Spectrum/or PFR Since increased sensitivity to 470-nm light can be used as a n assay for Ptt absorption and conversion to Pr~, a decrease in sensitivity can likewise indicate PFR absorption and conversion to P~. Thus the procedure for determining the U V absorption region for P?tt was to irradiate the cells for 1.5 min with 620-nm light (average intensity 6.45 • 1015 hv cm -2 s-l), followed b y a 3-rain irradiation with selected U V wavelengths set at approximately equal quantal levels (e.g., 350 n m = 8.0 • 10x5hv cm -2 s-l). The cells were left in darkness 1 rain and then the threshold to 470 n m was determined. The control experiment of 1.5 rain 620 nm, followed b y 4 rain in darkness showed an average threshold for a stop response at 0.62 • 1015 hv cm -2 s-z. Fig. 3 demonstrates t h a t the greatest loss in sensitivity followed irradiation with 390, 380, 370, and 360 nm. N o loss of sensitivity at all
Phototaxis in a Dinoflagellate
173
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followed e x p o s u r e to 320 n m which is in a g r e e m e n t with t h e results in Fig. 2. F o r comparison, t h e visible p o r t i o n of t h e t ~ a c t i o n s p e c t r u m (from F o r w a r d a n d D a v e n p o r t , 1968) is also p l o t t e d in Fig. 3.
4. Comparison o] Conversion o/PR to P~R by R and UV Light P r a t t a n d Briggs (1966) r e p o r t t h a t t h e in-vivo r a t i o of effectiveness of R to U V for this conversion of P ~ to PFR is a b o u t 100:1. I n Gyrodinium t h i s conversion increases s e n s i t i v i t y to 470 nm. Thus b y c o m p a r i n g t h e s e n s i t i v i t y t o this w a v e l e n g t h a f t e r i r r a d i a t i o n of t h e p h y t o c h r o m e w i t h different intensities of R (620-nm) a n d U V (320-nm, Fig. 2) light, it is possible to m e a s u r e t h e r e l a t i v e effectiveness of conversion. The level of responsiveness to 470 n m resulting from t h e P ~ t o PF~ conversion is m o s t q u a n t i t a t i v e l y m e a s u r e d b y i r r a d i a t i n g t h e P ~ after t h e positive response is abolished b y a 4-min exposure to 470 n m ( F o r w a r d a n d D a v e n p o r t , 1968). T h u s t h e p r o c e d u r e was to i r r a d i a t e t h e cells
174
R.B. Forward, Jr. :
with 470-nm (average intensity 12.8 • 1015 h~ em -2 S-1) for 4 rain, and then follow with a 3-min exposure to either 620 or 320 nm set at different intensity levels. After 1 min in darkness the threshold for a positive stop response to 470 nm was determined. In both cases, as seen in Fig. 4, the response saturates at high light intensities; below that there is a narrow range where the logarithm of response threshold is inversely proportional to the logarithm of the intensity of the sensitizing light. Below the lowest intensities of 620 and 320 nm shown in the figure responsiveness to 470 nm at the highest stimulation intensity was not observed. The ratio of the intensities at 320 and 620 nm needed to reach an arbitrary threshold value for 470-nm light is about 4:1 (at 3.0 • 1015 h~ em J' s-l), which certainly does not approach the 100 : 1 value reported by Pratt and Briggs (1966). Discussion
Two light receptive pigments appear to be involved in the stop response by Gyrodinium. The action spectra for sensitization and desensitization by phytochrome (Figs. 2, 3) are very different from those for the stop response (Fig. 1) in both the UV and visible regions. This strongly indicates the participation of two separate pigments. Haupt (1971) also suggests an interaction of two pigments for light induced chloroplast movements in another alga Mougeotia, although in this case both pigments can induce a behavioral response. By comparing the action spectrum for the stop response by Gyrodi. nium after an R irradiation (Fig. 1 a) with action spectra for other phytoflagellates, it is possible to determine the probable identity of the photoreceptor pigment. The loss of responsiveness at longer wavelengths above 530 nm and the large peak at 470 nm (Fig. 1 a) resemble responses of other dinoflagellates (Peridinium trochoidium and Gonyaulax catenella ; Halldal, 1958), of Euglena gracilis (Diehn, 1969) and of golvocales (Halldal, 1961). The remainder of the visible and near-UV action spectrum shows only two possible but not significant shoulders at 430 and 370 nm, and resembles that found for Platymonas, a member of the Volvocales (Halldal, 1961). An additional pronounced maximum occurs at about 365-375 nm in action spectra for phototaxis in Euglena (Diehn, 1969) and for chloroplast movements in the cMorophyeean alga Vaucheria (Itaupt and SchSnbohn, 1970); this is considered supporting evidence for a flavin as the photoreceptor pigment. Since such a UV peak is absent from the action spectrum for Platymonas ttalldal (1961) suggests that the pigment is a carotenoid. The action spectrum for Gyrodinlum also lacks a near-UV
Phototaxis in a Dinoflagellate
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maximum, so a carotenoid is likewise the probable photoreceptor. The strong response at 280 nm (Fig. 1 a) resembles that by Platymonas and probably corresponds to absorption by aromatic amino acids associated with a protein (Halldal, 1961), suggesting that the photoreceptor pigment in Gyrodinium is a carotenoprotcin. Pigments of this type are known to be present in a related dinoflagellate, Gyrodinium resplendens, in which the major carotenoids are /~-carotene, peridinin, dinoxanthin and diadinoxanthin (Loeblich and Smith, 1968). Further experiments, however, are necessary to identify the responsible carotenoid in G. dorsum. If a carotenoprotein is responsible for phototactic light reception following 1% irradiation, what happens after exposure to F R light ? In this ease the overall sensitivity is reduced, the blue peak shifts from 470 to 490 nm, the strong peak at 280 nm ("protein absorption") is lost, and a new maximum appears at 300-310 nm. An attractive explanation for this altered action spectrum is a trans to cis isomer shift by the carotenoid photoreeeptor. The characteristics of such a shift in isolated carotenoids are (1) lowering of the extinction coefficient, (2) shifting of the absorption maxima in the visible light, and (3) appearance of an absorption maximmn in the UV region (Weedon, 1965). All of these characteristics are observed after FR irradiation. However, if such a trans to ci8 isomer shift is in fact occurring, it must result in a dissociation of the carotenoid from the protein, since responsiveness to 280 nm disappears. Absorption maxima for the phytochrome in Gyrodinium are at shorter wavelengths than those found in higher plants (Butler et al., 1964). This finding appears typical for algae. Considering only Pa, isolated phytochrome from the green alga Mesotaenium had maximum absorption at 649 nm (Taylor and Bonner, 1967), and in a blue-green alga a pigment resembling phytochrome had Pg absorption at 520 nm (Scheibe, 1972). Finally, in a dinoflagellate, a representative of brown-colored algae, Forward and Davenport (1968) concluded from an action spectrum that the absorption maximum in the R was at 620 nm. Thus, in all detailed studies on algae, the red absorption maximum was found at wavelengths shorter than the 660-665-nm maximum of higher plants (Butler et al., 1964). In the UV region the PR of Gyrodinium has its absorption maximum at 320 nm, as compared to 370 nm for higher plants (Butler et al., 1964). An important question is whether this represents the true P~ absorption maximum or whether it results from shading by the phototactic pigment. Support for 320 nm as the real maximum is that the greatest sensitivity to 470-nm light does occur after exposure to this wavelength (Figs. 2, 3). Also some shift in absorption to lower wavelengths is expected from the 12a
l~lanta (Berl.), Bd. 111
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R.B. Forward, Jr.:
PR maximum at 620 nm. As compared to higher plants (Butler et al., 1964) this visible maximum represents a shift of 40-50 nm to shorter wavelengths. The UV maximum for PR in these plants occurs at 370 nm, and a shift of 40-50 nm places the maximum at 320-330 nm. Arguments for the P~ action spectrum resulting from shading by the phototactic pigment center on the fact that some of the UV wavelengths active in phytochromc conversion overlap those to which Gyrodinium can respond behaviorally. Since prolonged irradiation with 470 nm reduces sensitivity to this wavelength, prolonged irradiation with other behaviorally effective wavelengths can likewise reduce overall phototactic sensitivity. Thus illumination with UV potentially produces two opposing processes: (1) decreased behavioral sensitivity to blue (470-nm) light because the pigment involved in phototaxis is being irradiated, and (2) increased behavioral sensitivity because P~ is being converted to PF~. The action spectrum may then represent a compromise between these two processes. While growing under white light, however, both the phytoehrome and phototactic pigment are being simultaneously irradiated, and yet upon placing these cells in darkness, they show maximal phototactic sensitivity (Hand et al., 1967). Subsequently, total responsiveness after concurrent irradiation of both pigments is possible, and the foregoing evidence is most compelling for 320 nm as the true maximum for P~. Supression of responsiveness to 470 nm was used as the assay for the PF~ maximum in the UV (Fig. 3). In this experiment greatest stop response sensitivity occurs after irradiation with 320-nm light. This threshold is the same as that after 4 rain darkness (Fig. 3), indicating that the reponse is saturated, and not suppressed by the overall procedure. A uniformly high threshold is observed after irradiation with 360-390 nm. Since the visible P r a maximum (700 rim) is displaced about 30 nm from that for higher green plants, the expected UV peak should occur at about 30 nm below the normal 400-nm maximum for P ~ in higher plants (Butler, etal., 1964), namely at 370 nm. The observed broad peak encompasses this wavelength. The lack of responsiveness after irradiation with wavelengths greater than 400 nm might indicate interference by illumination of the phototactic pigment. The comparison of response activation (Px conversion to PF~) by the different quantal levels of UV and 1~ light allows an estimate of the relative effectiveness of conversions by these wavelengths. In the nonsaturated part of the curves (Fig. 4) the ratio of UV to R ranges around 4 : 1. This is certainly lower than the 100 : 1 ratio found by Pratt and Briggs (1966) and is closer to the 6:1 ratio shown for in-vitro phytochrome (Butler et al., 1964). There are at least two possible explanations for
Phototaxis in a Dinoflagellate
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this discrepancy. First the result m a y not be directly comparable, since P r a t t and Briggs (1966) were spectrophotometrically monitoring pigment concentration changes rather than a physiological response. This implies t h a t p h y t o c h r o m e conversions in light-grown cells leading to physiological events m a y be different from those in coleoptiles of darkgrown corn seedlings. Secondly, the 4:1 ratio does resemble t h a t reported for conversion b y UV irradiation of the p h y t o c h r o m e protein versus R irradiation of P a (Pratt and Butler, 1970). Since 320 n m is close to the protein absorption m a x i m u m at 280 nm, the lower ratio in Gyrodinium m a y reflect a composite absorption by the p h y t o c h r o m e ehromophore and protein. Thus the foregoing evidence strongly supports the involvement of a dual pigment system in phototaxis b y Gyrodinium, a carotenoprotein for the dh~ectional light response and a p h y t o c h r o m e which regulates the level of phototactic sensitivity b y the cells. I thank Dr. Beatrice Sweeney for her critical comments on the manuscript, and Meg Forward for her technical assistance. This study was supported by a Biomedical Science Support Grant from the National Institutes of Health to Duke University, and by a Major Grant from the Duke University Research Council. References Butler, W. L., Hendricks, S. B., Siegelman, H. W. : Action spectra of phytochi'ome in vitro. Photochem. Photobiol. 3, 521-529 (1964). Diehn, B. : Action spectra of phototactic responses in Euglena. Biochim. biophys. Acta (Amst.) 117, 136-143 (1969). Feinleib, M. E. H., Curry, G. M. : Methods for measuring phototaxis of cell populations and individual cells. Physiol. 20, 1083-1095 (1967). Feinlcib, IV[.E. H., Curry, G. M. : The relationship between stimulus intensity and oriented phototactic response (topotaxis) in Chlamydomonas. Physiol. Plant. 25, 346-357 (1971). Forward, R. B., Jr.: Changes in the photoresponse action spectrum of the dinoflagellate Gyrodinium dorsum Kofoid by red and far-red light. Planta (Berl.) 92, 248-258 (1970). Forward, R. B., Jr., Davenport, D.: Red and far-red light effects on a short-term behavioral response of a dinoflagellate. Science 161, 1028-1029 (1968). I-Ialldal, P. : Action spectra of phototaxis and related problems in Volvocales, Ulva
gametes and Dinophyceae. Physiol. Plant. 11, 118-153 (1958). HMldal, P.: Ultraviolet action spectra of positive and negative phototaxis in Platymonas subcordin/ormis. Physiol. Plant. 14, 133-139 (1961). Hand, W. G., Forward, R., Davenport, D.: Short-term photic regulation of a receptor mechanism in a dinofl~gellate. Biol. Bull. 133, 150-165 (1967). Haupt, W.: Schwachlichtbewegung des Mougeotia-Chloroplasten im Blaulicht. Z. Pflanzenphysiol. 65, 248-265 (1971). i2b
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R . B . Forward, Jr. : Phototaxis in a Dinoflagellate
Haupt, W., Sch6nbohn, E.: Light oriented chloroplast movements. In: Photobiology of microorganisms, p. 283-308 (Halldal, P., ed.). New York: Wiley-In,rscience 1970. Huth, K. : Bewegung und Orienticrung bei Volvox aureus. Ehrb. Z. Pflanzenphysiol. 62, 436 450 (1970). Loeblich, A. R., HI, Smith, V. E.: Chloroplast pigments of the marine dinoflagellate Gyrodinium resplendens. Lipids 8, 5-13 (1968). Pratt, L. It., Briggs, W. R.: Phytochrome and non-photochemical reactions of phytochrome in vivo. Plant Physiol. 41, 467474 (1966). Pratt, L. H., Butler, W. L. : Phytochrome conversion by ultraviolet light. Photochem. Photobiol. 11, 503-509 (1970). Scheibe, J.: Photoreversible pigment occurrence in a blue-green alga. Science 176, 1037-1039 (1972). Taylor, A. 0., Bonner, B. A.: Isolation of phytochrome from the alga Mesotaenium and liverwort Sphaerocarpos. Plant Physiol. 42, 762-766 (1967). Weedon, B. C. L. : Chemistry of the carotenoids. In: Chemistry and biochemistry of plant pigments, p. 75-126 (Goodwin, T.W., ed.). New York: Acad. Press 1964.
Phototaxis in a Dinoflagellate: Action Spectra as Evidence for a Two-Pigment System* R i c h a r d B. F o r w a r d , Jr. Zoology Department, Duke University Marine Laboratory, Beaufort, North Carolina 28516, U.S.A. Received October 5, 1972 / January 31, 1973
Summary. Action spectra were determined in the UV region of the spectrum for the first phase of the phototactie response (stop response) and for the phytochrome pigment associated with this response in the dinoflageltate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after F R irradiation they shift to 490 and 300-310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of PI~ in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The PFR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of PI~ to PFR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.
Introduction The dinoflagellate Gyrodinium dorsum K o f o i d r e s p o n d s in a p h o t o t a c t i c m a n n e r t o light s t i m u l a t i o n . A t high light intensities t h e response sequence consists of a n initial cessation of m o v e m e n t (stop response) followed b y s w i m m i n g in t h e d i r e c t i o n of t h e s t i m u l u s b e a m ( H a n d et al., !967). A similar, i n i t i a l cessation of m o v e m e n t u p o n s t i m u l a t i o n with l i g h t occurs in o t h e r p h y t o f l a g e l l a t e s , e. g., Chlamydomonas (Feinlieb a n d Curry, 1971) a n d Volvox ( I t u t h , 1970). I n all cases this s t o p response is considered a n i n t e g r a l p a r t of t h e p h o t o t a e t i c response, a n d t h u s i t can be used as a n i n d i c a t o r of p h o t o t a c t i c light p e r c e p t i o n . I n t h e visible region Gyrodinium is m o s t responsive to blue light; however, t h e exac~ m a x i m u m d e p e n d s u p o n w h e t h e r t h e cells are pre-
* Abbreviations. F R ~far-red, R ~ red, PFl~=far-red-absorbing form of phytochrome, P~=red-absorbing form of phytochrome, UV=ultraviolet.
168
R . B . Forward, Jr. :
i r r a d i a t e d with R (620-nm) or F R (700-nm) light, a n d these effects of long-wave light are m u t u a l l y reversible ( F o r w a r d , 1970). This d e p e n d e n c e u p o n R a n d F R light a n d o t h e r evidence ( F o r w a r d a n d D a v e n p o r t , 1968) i n d i c a t e t h a t a p h y t o c h r o m e p i g m e n t is i n v o l v e d as a n a d d i t i o n a l f a c t o r in p h o t o t a x i s . A t e n t a t i v e m o d e l for t h e p h o t o t a e t i e p i g m e n t s y s t e m p r o p o s e d t h e i n t e r a c t i o n of two pigments, a n u n i d e n t i f i e d b l u e - a b s o r b i n g p i g m e n t for t h e directional response a n d a p h y t o c h r o m e controlling t h e s e n s i t i v i t y of t h e cells ( F o r w a r d , 1970). Since all p r e v i o u s studies w i t h Gyrodinium were c o n d u c t e d w i t h visible light, i t was t h o u g h t t h a t f u r t h e r i n f o r m a t i o n a b o u t t h e p i g m e n t s i n v o l v e d m i g h t be gained b y determining t h e a c t i o n s p e c t r a for b o t h t h e b l u e - a b s o r b i n g a n d t h e phy~ochrome p i g m e n t in the U V region. Such a n e x a m i n a t i o n is t h e s u b j e c t of the p r e s e n t investigation.
Methods and Materials The culture methods for Gyrodinium dorsum Kofoid remain as previously described (Hand et al., 1967). Cells were grown in a model CEL-44 growth chamber (Sherer Gillet, Marshall, Mich., U.S.A.) with a light-dark cycle of 16:8 h and at a temperature of 19~ Since responsiveness varies with culture age, only 6-d-old cultures were used, and all tests were begun 3 h after the onset of the light period. The equipment for monitoring photoresponses of individual cells resembles that described by Feinleib and Curry (1967). Cells are observed in dark field under a model UPL inverted microscope (Karl Zeiss, New York, N. Y.). The dark field illumination light is filtered with either a 621 or a 701-nm interference filter (Optics Technology, Palo Alto, Calif., U.S.A.). The light stimulus source is a model f/3.5 monochromator (Farrand Optical Co., Mount Vernon, N.Y., U.S.A.), coupled to a 150-W Xenon arc lamp. Except where noted, the full band pass for stimulus wavelengths is 10 nm. This light is focused onto the experimental vessel (florimeter cell made of infrasil quartz; interior dimensions 1 0 x 5 x 5 mm) by means of quartz lenses (Oriel Optics Corp., Stamford, Conn., U.S.A.), and its spectral purity regulated by Coming filters (Coming Glass Works, Coming, N. Y., U.S.A.), namely for the UV region, No. 7-54; for the blue, No. 4-96; and for the yellow-red region, No. 3-67. Since the arc position of the lamp varied with each experiment, intensities of the experimental wavelengths were measured before each experiment, using a model 65A radiometer (Yellow Springs Instrument Co., Yellow Springs, O., U.S.A.). Stimulus duration is controlled by a Uniblitz electromagnetic shutter (Vincent Associates, Rochester, N.Y., U.S.A.) which is set at 1.2 s. The intensity of the stimulus light is controlled by neutral density filters. In all experiments the initial stop response is used as the index of photoresponsiveness. Recording the responses was accomplished by photographing through the microscope with a 35-ram camera using Kodak-2475 recording film. Since the response latency is about 0.4 s, the solenoid for triggering the camera was activated 0.4 s after the beginning of light stimulation, as timed by an electronic timing circuit. By using a 0.5-s exposure, responding cells can be easily differentiated from swimming cells on the photographic negative, and the percent response calculated.
Pho~otaxis in a DinofiagelIate
169
Since the microscope dark-field-illumination light can potentially alter cell responsiveness, this illumination only occurred while the ceils were actually being stimulated and responses photographed. At all other times the microscope illumination light was prevented from irradiating the cells by the appropriate placement of light shields. This light was filtered with a 601-nm interference filter for the determination of the stop response action spectrum after R irradiation, and with a 701-nm filter in all other experiments. The response criteria remain as previously established (Forward and Davenport, 1968). A response in which more than 50 % of the cells cease moving upon stimulation is designated as a "positive stop response". In all experiments the lowest stimulus light intensity (threshold) which would initiate a positive stop response was used to measure phototactie sensitivity under different conditions. Since the exact procedure of pre-illumination and stimulation varied with each experiment, they will be described in detail in the following section.
Results
1. Action Spectra/or the Stop Response Following R and F R Irradiation The visible action spectrum for the positive stop response depends upon whether the cells are pre-irradiated with 1%(620-nm) or F R (700-nm) light (Forward, 1970). These action spectra were redetermined to include wavelengths in the UV, utilizing the following procedures. To convert the p h y t o c h r o m e to P~a, aliquot cell samples were r e m o v e d from the culture chamber and irradiated for I min with 620-nm light (average intensity of 4.92 • 10 z5 hv cm -~ s-Z). The cells were then left in darkness for 1 min after which t h e y were stimulated with either an increasing or decreasing sequence of intensities at selected wavelengths in the visible and UV. The lowest intensity at which a positive stop response could be elicited was designated as the threshold intensity for t h a t wavelength. Since sensitivity is k n o w n to change over time in darkness, the order of stimulus intensity presentation (increasing or decreasing) was alternated from one trial to the next. However, the observed thresholds for each procedure were usually identical. A new cell sample was used for each determination at each wavelength. Fig. I a shows t h a t response m a x i m a occur at 470 and 280-nm. To convert the p h y t o c h r o m e to P~, cells were irradiated with 620-nm light (average intensity 5.5 • 10 z5 hv cm -2 s -z) for 1.5 rain, followed b y a 700-nm irradiation (full b a n d pass 20-nm; average intensity 6.45 • 10 z5 hv cm -2 s -z) for the same time. The cells were then left in darkness for 1 rain, a n d the threshold for a positive response at the different wavelengths determined as described above. Fig. 1 b shows t h a t the overall sensitivity to light is reduced b y F R light, and the m a x i m a are shifted to 490 and 300-310 nm. The UV m a x i m u m was placed at 300-310 n m
170
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because the lowest percentage of t o t a l l y u n r e s p o n s i v e cultures was f o u n d u p o n s t i m u l a t i o n with these wavelengths. I t should also be n o t e d t h a t after the R - F R irradiations no response to 280 n m was observed.
2. UV Action Spectrum/or Pi~ Responsiveness to 470 n m can be abolished b y a 4 m i n exposure to this wavelength. However, the response can be r e a c t i v a t e d b y s u b s e q u e n t
Phototaxis in a Dinoflagellate
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conversion of Pa to PFg by exposure to I~ light (Forward and Davenport, 1968). The level of the returned sensitivity to blue (470 nm) light varies with magnitude of phytochrome conversion from Pit to PFg and therefore can be used as a sensitive indicator of conversion (Forward, 1970). Thus in this experiment the UV action spectrum for P~ is determined by measuring the reactivation of responsiveness to 470 nm by selected UV wavelengths after a prolonged blue exposure. The procedure was to abolish responsiveness to blue (470 nm) by a 4-rain exposure to this light (average intensity 12.4 • 1015 hv cm -a s-l). This was followed by a 3-rain exposure to selected wavelengths between 410 and 310 nm, set at approximately equal quantal levels (e.g., 350 nm ----8.7 • 10 Is hv cm -~ s-X). Then after 1 rain in darkness the threshold for a positive stop response to 470 nm was determined. Controls demonstrated that following the initial 4-rain blue irradiation a n d 4 rain in darkness, t h e level of s t o p p i n g u p o n s t i m u l a t i o n was t h a t e x p e c t e d for a r a n d o m response (20-35 %). P r o l o n g e d exposure to 290 a n d 280 n m was n o t possible since t h e cells d i e d a l m o s t i m m e d i a t e l y . Fig. 2 shows t h a t t h e a p p a r e n t p e a k for P ~ a b s o r p t i o n in t h e n e a r U V is a t 320 nm, since g r e a t e s t s e n s i t i v i t y to 470 n m occurs a f t e r i r r a d i a t i o n
172
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with this wavelength. To completely illustrate the action spectrum for P~, the visible portion (from F o r w a r d and Davenport, 1968) is plotted on the right of Fig. 2.
3. UV Action Spectrum/or PFR Since increased sensitivity to 470-nm light can be used as a n assay for Ptt absorption and conversion to Pr~, a decrease in sensitivity can likewise indicate PFR absorption and conversion to P~. Thus the procedure for determining the U V absorption region for P?tt was to irradiate the cells for 1.5 min with 620-nm light (average intensity 6.45 • 1015 hv cm -2 s-l), followed b y a 3-rain irradiation with selected U V wavelengths set at approximately equal quantal levels (e.g., 350 n m = 8.0 • 10x5hv cm -2 s-l). The cells were left in darkness 1 rain and then the threshold to 470 n m was determined. The control experiment of 1.5 rain 620 nm, followed b y 4 rain in darkness showed an average threshold for a stop response at 0.62 • 1015 hv cm -2 s-z. Fig. 3 demonstrates t h a t the greatest loss in sensitivity followed irradiation with 390, 380, 370, and 360 nm. N o loss of sensitivity at all
Phototaxis in a Dinoflagellate
173
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followed e x p o s u r e to 320 n m which is in a g r e e m e n t with t h e results in Fig. 2. F o r comparison, t h e visible p o r t i o n of t h e t ~ a c t i o n s p e c t r u m (from F o r w a r d a n d D a v e n p o r t , 1968) is also p l o t t e d in Fig. 3.
4. Comparison o] Conversion o/PR to P~R by R and UV Light P r a t t a n d Briggs (1966) r e p o r t t h a t t h e in-vivo r a t i o of effectiveness of R to U V for this conversion of P ~ to PFR is a b o u t 100:1. I n Gyrodinium t h i s conversion increases s e n s i t i v i t y to 470 nm. Thus b y c o m p a r i n g t h e s e n s i t i v i t y t o this w a v e l e n g t h a f t e r i r r a d i a t i o n of t h e p h y t o c h r o m e w i t h different intensities of R (620-nm) a n d U V (320-nm, Fig. 2) light, it is possible to m e a s u r e t h e r e l a t i v e effectiveness of conversion. The level of responsiveness to 470 n m resulting from t h e P ~ t o PF~ conversion is m o s t q u a n t i t a t i v e l y m e a s u r e d b y i r r a d i a t i n g t h e P ~ after t h e positive response is abolished b y a 4-min exposure to 470 n m ( F o r w a r d a n d D a v e n p o r t , 1968). T h u s t h e p r o c e d u r e was to i r r a d i a t e t h e cells
174
R.B. Forward, Jr. :
with 470-nm (average intensity 12.8 • 1015 h~ em -2 S-1) for 4 rain, and then follow with a 3-min exposure to either 620 or 320 nm set at different intensity levels. After 1 min in darkness the threshold for a positive stop response to 470 nm was determined. In both cases, as seen in Fig. 4, the response saturates at high light intensities; below that there is a narrow range where the logarithm of response threshold is inversely proportional to the logarithm of the intensity of the sensitizing light. Below the lowest intensities of 620 and 320 nm shown in the figure responsiveness to 470 nm at the highest stimulation intensity was not observed. The ratio of the intensities at 320 and 620 nm needed to reach an arbitrary threshold value for 470-nm light is about 4:1 (at 3.0 • 1015 h~ em J' s-l), which certainly does not approach the 100 : 1 value reported by Pratt and Briggs (1966). Discussion
Two light receptive pigments appear to be involved in the stop response by Gyrodinium. The action spectra for sensitization and desensitization by phytochrome (Figs. 2, 3) are very different from those for the stop response (Fig. 1) in both the UV and visible regions. This strongly indicates the participation of two separate pigments. Haupt (1971) also suggests an interaction of two pigments for light induced chloroplast movements in another alga Mougeotia, although in this case both pigments can induce a behavioral response. By comparing the action spectrum for the stop response by Gyrodi. nium after an R irradiation (Fig. 1 a) with action spectra for other phytoflagellates, it is possible to determine the probable identity of the photoreceptor pigment. The loss of responsiveness at longer wavelengths above 530 nm and the large peak at 470 nm (Fig. 1 a) resemble responses of other dinoflagellates (Peridinium trochoidium and Gonyaulax catenella ; Halldal, 1958), of Euglena gracilis (Diehn, 1969) and of golvocales (Halldal, 1961). The remainder of the visible and near-UV action spectrum shows only two possible but not significant shoulders at 430 and 370 nm, and resembles that found for Platymonas, a member of the Volvocales (Halldal, 1961). An additional pronounced maximum occurs at about 365-375 nm in action spectra for phototaxis in Euglena (Diehn, 1969) and for chloroplast movements in the cMorophyeean alga Vaucheria (Itaupt and SchSnbohn, 1970); this is considered supporting evidence for a flavin as the photoreceptor pigment. Since such a UV peak is absent from the action spectrum for Platymonas ttalldal (1961) suggests that the pigment is a carotenoid. The action spectrum for Gyrodinlum also lacks a near-UV
Phototaxis in a Dinoflagellate
175
maximum, so a carotenoid is likewise the probable photoreceptor. The strong response at 280 nm (Fig. 1 a) resembles that by Platymonas and probably corresponds to absorption by aromatic amino acids associated with a protein (Halldal, 1961), suggesting that the photoreceptor pigment in Gyrodinium is a carotenoprotcin. Pigments of this type are known to be present in a related dinoflagellate, Gyrodinium resplendens, in which the major carotenoids are /~-carotene, peridinin, dinoxanthin and diadinoxanthin (Loeblich and Smith, 1968). Further experiments, however, are necessary to identify the responsible carotenoid in G. dorsum. If a carotenoprotein is responsible for phototactic light reception following 1% irradiation, what happens after exposure to F R light ? In this ease the overall sensitivity is reduced, the blue peak shifts from 470 to 490 nm, the strong peak at 280 nm ("protein absorption") is lost, and a new maximum appears at 300-310 nm. An attractive explanation for this altered action spectrum is a trans to cis isomer shift by the carotenoid photoreeeptor. The characteristics of such a shift in isolated carotenoids are (1) lowering of the extinction coefficient, (2) shifting of the absorption maxima in the visible light, and (3) appearance of an absorption maximmn in the UV region (Weedon, 1965). All of these characteristics are observed after FR irradiation. However, if such a trans to ci8 isomer shift is in fact occurring, it must result in a dissociation of the carotenoid from the protein, since responsiveness to 280 nm disappears. Absorption maxima for the phytochrome in Gyrodinium are at shorter wavelengths than those found in higher plants (Butler et al., 1964). This finding appears typical for algae. Considering only Pa, isolated phytochrome from the green alga Mesotaenium had maximum absorption at 649 nm (Taylor and Bonner, 1967), and in a blue-green alga a pigment resembling phytochrome had Pg absorption at 520 nm (Scheibe, 1972). Finally, in a dinoflagellate, a representative of brown-colored algae, Forward and Davenport (1968) concluded from an action spectrum that the absorption maximum in the R was at 620 nm. Thus, in all detailed studies on algae, the red absorption maximum was found at wavelengths shorter than the 660-665-nm maximum of higher plants (Butler et al., 1964). In the UV region the PR of Gyrodinium has its absorption maximum at 320 nm, as compared to 370 nm for higher plants (Butler et al., 1964). An important question is whether this represents the true P~ absorption maximum or whether it results from shading by the phototactic pigment. Support for 320 nm as the real maximum is that the greatest sensitivity to 470-nm light does occur after exposure to this wavelength (Figs. 2, 3). Also some shift in absorption to lower wavelengths is expected from the 12a
l~lanta (Berl.), Bd. 111
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R.B. Forward, Jr.:
PR maximum at 620 nm. As compared to higher plants (Butler et al., 1964) this visible maximum represents a shift of 40-50 nm to shorter wavelengths. The UV maximum for PR in these plants occurs at 370 nm, and a shift of 40-50 nm places the maximum at 320-330 nm. Arguments for the P~ action spectrum resulting from shading by the phototactic pigment center on the fact that some of the UV wavelengths active in phytochromc conversion overlap those to which Gyrodinium can respond behaviorally. Since prolonged irradiation with 470 nm reduces sensitivity to this wavelength, prolonged irradiation with other behaviorally effective wavelengths can likewise reduce overall phototactic sensitivity. Thus illumination with UV potentially produces two opposing processes: (1) decreased behavioral sensitivity to blue (470-nm) light because the pigment involved in phototaxis is being irradiated, and (2) increased behavioral sensitivity because P~ is being converted to PF~. The action spectrum may then represent a compromise between these two processes. While growing under white light, however, both the phytoehrome and phototactic pigment are being simultaneously irradiated, and yet upon placing these cells in darkness, they show maximal phototactic sensitivity (Hand et al., 1967). Subsequently, total responsiveness after concurrent irradiation of both pigments is possible, and the foregoing evidence is most compelling for 320 nm as the true maximum for P~. Supression of responsiveness to 470 nm was used as the assay for the PF~ maximum in the UV (Fig. 3). In this experiment greatest stop response sensitivity occurs after irradiation with 320-nm light. This threshold is the same as that after 4 rain darkness (Fig. 3), indicating that the reponse is saturated, and not suppressed by the overall procedure. A uniformly high threshold is observed after irradiation with 360-390 nm. Since the visible P r a maximum (700 rim) is displaced about 30 nm from that for higher green plants, the expected UV peak should occur at about 30 nm below the normal 400-nm maximum for P ~ in higher plants (Butler, etal., 1964), namely at 370 nm. The observed broad peak encompasses this wavelength. The lack of responsiveness after irradiation with wavelengths greater than 400 nm might indicate interference by illumination of the phototactic pigment. The comparison of response activation (Px conversion to PF~) by the different quantal levels of UV and 1~ light allows an estimate of the relative effectiveness of conversions by these wavelengths. In the nonsaturated part of the curves (Fig. 4) the ratio of UV to R ranges around 4 : 1. This is certainly lower than the 100 : 1 ratio found by Pratt and Briggs (1966) and is closer to the 6:1 ratio shown for in-vitro phytochrome (Butler et al., 1964). There are at least two possible explanations for
Phototaxis in a Dinoflagellate
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this discrepancy. First the result m a y not be directly comparable, since P r a t t and Briggs (1966) were spectrophotometrically monitoring pigment concentration changes rather than a physiological response. This implies t h a t p h y t o c h r o m e conversions in light-grown cells leading to physiological events m a y be different from those in coleoptiles of darkgrown corn seedlings. Secondly, the 4:1 ratio does resemble t h a t reported for conversion b y UV irradiation of the p h y t o c h r o m e protein versus R irradiation of P a (Pratt and Butler, 1970). Since 320 n m is close to the protein absorption m a x i m u m at 280 nm, the lower ratio in Gyrodinium m a y reflect a composite absorption by the p h y t o c h r o m e ehromophore and protein. Thus the foregoing evidence strongly supports the involvement of a dual pigment system in phototaxis b y Gyrodinium, a carotenoprotein for the dh~ectional light response and a p h y t o c h r o m e which regulates the level of phototactic sensitivity b y the cells. I thank Dr. Beatrice Sweeney for her critical comments on the manuscript, and Meg Forward for her technical assistance. This study was supported by a Biomedical Science Support Grant from the National Institutes of Health to Duke University, and by a Major Grant from the Duke University Research Council. References Butler, W. L., Hendricks, S. B., Siegelman, H. W. : Action spectra of phytochi'ome in vitro. Photochem. Photobiol. 3, 521-529 (1964). Diehn, B. : Action spectra of phototactic responses in Euglena. Biochim. biophys. Acta (Amst.) 117, 136-143 (1969). Feinleib, M. E. H., Curry, G. M. : Methods for measuring phototaxis of cell populations and individual cells. Physiol. 20, 1083-1095 (1967). Feinlcib, IV[.E. H., Curry, G. M. : The relationship between stimulus intensity and oriented phototactic response (topotaxis) in Chlamydomonas. Physiol. Plant. 25, 346-357 (1971). Forward, R. B., Jr.: Changes in the photoresponse action spectrum of the dinoflagellate Gyrodinium dorsum Kofoid by red and far-red light. Planta (Berl.) 92, 248-258 (1970). Forward, R. B., Jr., Davenport, D.: Red and far-red light effects on a short-term behavioral response of a dinoflagellate. Science 161, 1028-1029 (1968). I-Ialldal, P. : Action spectra of phototaxis and related problems in Volvocales, Ulva
gametes and Dinophyceae. Physiol. Plant. 11, 118-153 (1958). HMldal, P.: Ultraviolet action spectra of positive and negative phototaxis in Platymonas subcordin/ormis. Physiol. Plant. 14, 133-139 (1961). Hand, W. G., Forward, R., Davenport, D.: Short-term photic regulation of a receptor mechanism in a dinofl~gellate. Biol. Bull. 133, 150-165 (1967). Haupt, W.: Schwachlichtbewegung des Mougeotia-Chloroplasten im Blaulicht. Z. Pflanzenphysiol. 65, 248-265 (1971). i2b
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