~2-6989J92 %S.OO + 0.00 Copyright 0 1992 Pergamon Press Ltd
Vision Res. Vol. 32, NO. 9, pp. lS93-1600, 1992 Printed in Great Britain. All rights reserved
The Eyespot of Chlamydomonas reinhardtii: a Comparative Microspectrophotometric Study F. CRESCTTELLI,**
T. W. JAMES,* J. M. ERICKSON,*
E. R. LOEW,? W. N. McFARLANDS
Received 5 August 1991; in revisedform 4 MarCh 1992
The eyespot of C~l~y~~nus reinhardtii is believed to utilize a rhodopsin-like pigment in its responses to light. This paper examines its eyespot by means of microspectrophotometry with the finding of an absorption spectrum with two bands, an A-band in the blue, and a B-band in the green. This spectrmn is identical to that pre~ously recorded from the eyespot of Eugha grudis. AS with Eugienu the B-band was found to have dichroic character and its spectrum was similar to the absorption curve of rhodopsin. This A-B-spectrum was always recorded from a single granule in each cell. It is concluded that both E. gracifis and C. r&&r&ii may uti%zea rhodopsin-like pigment as the photopigment associated with the eyespot response to light. In both these algae a few particles in each cell were found whose spectra consisted of two other bands, C and D, blue- and red-shifted, respectively, relative to the eyespot A-B-bands. There is some reason to believe that the C-D-granules may also be involved in certain ~ght~on~olled activities of the cells. ChIamydomonas Eyespot
Rhodopsin
Spectral absorption
INTRODUCTION In the preceding paper (James, Crescitelli, Loew & McFarland, 1992) the spectral absorbance of individual eyespots of EuglePla gracilis was shown to consist of two absorption bands? one in the blue (A-band), the second in the green (B-band). Unlike the A-band, the B-band displayed dichroic character, a property that suggested the absorption of a pigment oriented in the anisotropic, quasi-crystalline paraflagellar body (PFB). The isotropic A-band was tentatively interpreted to be the absorption of a pigment in the orange-red stigma in close association with the PFB. A significant feature of this study was the similarity of the B-band to the spectral absorption curve of rhodopsin. Because of this finding attempts were made to obtain chemical evidence of a rhodopsinlike system in the eyespot but these were unsuccessful because photic bleaching did not occur so that the possible effects of hydroxylamine and of added retinal could not be examined. This idea of a rhodopsin-like photosystem in phototactic algae is not new because recent studies have reported chemical evidence that the eyespot of Chlamydomonas rein~~rdti~ does indeed possess such a photopigment. Taking advantage of mutant FN68 which is defective in the synthesis of retinal, Foster, Saranak, *Department of Biology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024-1606, U.S.A. tPhysiology Division of Biological Sciences and NYS College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. ICatalina Marine Science Center, University of Southern California, P.O. Box 396, Avalon, CA 90704, U.S.A. *Deceased.
Patel, Zarilli, Okabe, Kline and Nakanishi (1984) were able to demonstrate an increase in phototactic sensitivity by the addition of retinal and the resulting action spectrum was similar to the spectral absorption of bovine rhodopsin. Apparently, opsin was present in this mutant so that the addition of retinal restored the photopigment required for the light response. Along with our study of the Euglena eyespot (James et al., 1992) the evidence of a rhodopsin-like system in Chlamydomonas offers the possibility of advancing the subject in two directions by finding answers to two related questions: (a) what is the spectral absorbance of the Chlamydomonas eyespot and does it support the published chemical evidence of a retinal-based photopigment; and (b) what is the form of the Chlumydomonas eyespot spectrum? Is it like that of E. gracilis, with Aand B-bands, the latter band alone showing a dichroic property? If the answer to this second question .is positive it adds confidence to the hypothesis of a rhodopsin-like pigment in E. gracilis for which the necessary chemical evidence is lacking. A positive answer to both these questions would serve to further generalize the concept of an early phylogenetic origin of retinalbased pigments in biological photosensitivity. In this paper, therefore, we intend to present evidence that bears on both these questions. MATERIALS AND METHODS As already indicated (James et al., 1992), algal cells with chlorophyll offer complications in the recording of spectral absorbance in that the two main chlorophyll bands dominate the spectrum and make it difficult to
1593
F. CRESCITELLI
1594
sort out the eyespot response. For this reason Euglena cells were used that were bleached free of chlorophyll with streptomycin or by growth in the dark. Cells from these cultures yielded eyespot absorption curves free of chlorophyll contamination and provided data that were summarized in the Introduction. We also recorded the eyespot absorption of green cells of the Z-strain of E. gracilis and obtained the same A-B-spectrum, the Bband with similar dichroic behavior. This result was useful in showing that the A-B-spectrum is not the consequence of an impaired metabolism due to the deficiency of chlorophyll. For the present study, we used a chlorophyll-deficient mutant of C. reinhardtii, BF4/Ml8, initially obtained from J. Girard-Bascou and P. Bennoun. BF4/M18 is a double, nuclear mutant deficient in the light-harvesting chlorophyll complex associated with photosystem II (Olive et al., 1981) and lacking photosystem I (Girard et al., 1980). The spectral recordings were obtained with the same microspectrophotometer and procedures as in the Euglena study, already cited. The living cells, lightly compressed between two cover slips in a metal frame holder, were placed on the microscope stage and visualized on a video monitor which received the output of an infrared camera mounted on the exit optics of the microscope. A quiescent cell was selected, its eyespot was found with the field illuminated with light at 470 nm as in the Euglena study. On the monitor screen the Chlumy domonas eyespot was seen as a dark body on a lighter granular cytoplasmic background. Since bodies other than the eyespot were normally present, and their morphology was often not unlike that of the eyespot, it was necessary to distinguish these as elements different from the single eyespot in each cell. This was carried out by taking advantage of the eyespot color. The field illumination was set at 470 nm, changed to 560 nm and then
back to 470 nm, a flip-flop procedure that caused the eyespot to disappear from view and then to reappear. Granules other than the eyespot remained in view during this procedure. This technique did indeed pick out the eyespot. Only one granule in each cell responded positively to this 470-560-470 maneuver, and it was this granule, and not the others, from which the A--B spectrum to be described was obtained. Once the eyespot was located, the field was returned to infrared viewing and the light probe beam at 750 nm was turned on and adjusted to an appropriate dimension to fit the eyespot, the smallest being a square with 3 pm sides. The probe was then placed in the clear medium outside the cell and a spectral scan was made over the chosen wavelength span. The data were stored in the computer and the probe next positioned as close to the center of the selected eyespot as possible. A similar spectral scan was again made and the difference spectrum resulting from these two scans was viewed and then stored for later printing and study. The cells of Chlaml,domonas, though relatively small compared to those of Euglena, remained alive and motile for hours between the cover slips and we were able to collect some 300 recordings from about 200 different eyespots. The microspectrophotometer (MSP) was described in the previous paper and employed as there described. It was there described as an instrument capable of recording the correct spectra of a human red blood cell, a visual rod from the frog retina and of a didymium filter.
RESULTS It was quickly discovered that the granules that reacted to the flip-flop test yielded spectra characteristically different from those recorded from the granules that did not flip-flop. The former gave spectra (Fig. 1)
Wavelength FIGURE
1. Eyespot
spectra
showing
Y[ (I!
(nm)
A- and B-bands from E. gracilis (1) and C. reinhardtii are 0.0457 [curve (l)] and 0.0764 [curve (2)].
(2). The 100% normalized
0.D.s
CHLAMYDOMONAS
400
500
450 Wavelength
1595
EYESPO-I
550
600
(nm)
FIGURE 2. Spectral absorption of the two types of granules recorded from the C. reinhardtii eyespot. Curve (1) Granule that went out in 470-560 nm shift, showing A- and B-bands. Curve (2) Granule that remained in 47&560 nm shift, showing Cand D-bands. Granule 1 was an eyespot; granule 2 was next to eyespot. Normalized 0.D.s: 0.0871 [curve (l)], 0.0097 [curve (2)].
the A- and B-bands already known from the Euglena experiments, while the latter granules lacked these two bands, but not all were spectroscopically silent, for we consistently recorded from a few a double-banded spectrum consisting of what we call C- and D-bands shifted significantly from the locations of the A- and B-bands. As seen in Fig. 2, the C-band is blue-shifted by some 18-20 nm from the A-band, while the D-band is red-shifted by an even greater amount (33-35 nm) from the B-band. In addition, the form of the B- and
with
D-spectra differ in that the former is featureless while the latter is broader and seems to have a multi-banded structure (Fig. 2). The different spectral locations of the B- and D-bands explains why the eyespot disappeared in the flip from 470 to 560 nm while the granules responsible for the D-band persisted. Incidentally, after the discovery in Chlamydomonas of spots with C- and D-bands, we returned to E. gracilis and found similar bodies that remained in view in the 470-560-test and gave a spectrum with C- and D-bands (Fig. 3).
20-
0
FIGURE
“,“,,,,‘,,,,,,,,,,,,, 400
450 Wavelength
500 (nm)
550
600
3. The C- and D-bands from Euglena [curve (l)] and Chfumydomonm [curve (2)] from granules that remained in the 470-560-test. Normalized 0.D.s: 0.0435 [curve (l)], 0.0097 [curve (2)].
F. CRESCITELLI
1596
As in E. gracilis, the Chlamydomonas A-B spectrum was found in a small and highly localized spot, and movement of the probe away from this spot by as little as 1 ,~rn resulted in a reduction or disappearance of the spectrum, an example being that of Fig. 4 where a 3 ,um probe was first placed at a point of largest response [curve (l)] and then moved 1 pm away from this point [curve (2)]. This rather precise localization favors the idea of a pigment in a very small region of the cell as the source of the A-B-spectrum. It is important to point out that it was never possible to obtain records with the A-band alone or the B-band alone, a situation that we also encountered in our Euglena work. This does not necessarily disprove the idea that the A- and B-bands originate in different structures within the eyespot complex, since over dimensions of the order of 3 pm and more, light scattering probably prevents the precise localization of probe beam to only one of the two possible structures. Another resemblance to the Euglena spectrum was the effect of varying probe size. In the E. gracilis eyespot a small probe centered on the active region gave a spectrum in which the A- and B-bands were fused into a broadly-rounded maximum. Increasing probe size caused a decrease of spectrum magnitude and brought out the A- and B-bands. A similar response was found in Chlamydomonas (Fig. 5) where a probe with 3.0 pm sides yielded a spectrum [curve (I)] in which the two bands were not clearly distinguished. Increasing probe size to 4 pm [curve (2)], to 5 pm [curve (3)] and to 6 pm [curve (4)] led to a progressive separation of the two bands. In addition, the maximum (100%) O.D. values of these four recordings decreased progressively (0.1160, 0.0805, 0.0469, 0.0239). These two effects of probe beam size may be ascribed to a dilution of pigment concentration as the probe size increased to include
er ui
cytoplasmic areas outside the eyespot that iackcd pugment, although a possible effect of light scattering C;~IF not be discounted. Perhaps the most significant property of the c Mmzr domonas spectrum, as with E. gracilis, was the effect observed by a 90” change in polarization of the probe from a parallel to a perpendicular orientation with respect to the cell anterior- posterior axis. Compared to Euglena, the Chlamydomonas cell is smaller and rounder, so that determining the cell axis was difficult and uncertain. We therefore employed the location of the Ragellar swirl as indicative of the anterior pole of the cell. The result of this 90“ change in polarization axis was II selective reduction of the B-band. suggesting an anisotropic structure as the source of this absorption (Fig. 6). Figure 6, selected because of its relatively high dichroic ratio (2.2) is at the upper level of ratios obtained from eyespots, which show a considerable variance. In one series of recordings using the same eyespot and IO successive probings with the same 3.0pm probe, dichroic ratios from 1.30 to 1.92 were obtained. The variability was even greater when a comparison of different eyespots was made. This variability is perhaps to be expected when recording from living cells in which the eyespot preferred axis is changing constantly due to cytoplasmic movements. In any case, the result pictured in Fig. 6 along with the statistical data of Table 1 confirms the idea of an anisotropic structure as the source of the B-band in Chlamydomonas. As with Euglena no evidence for anisotropy was seen for the A-band (Table 1). From a biological point of view perhaps the most interesting similarity in the results with these two algal species is that the spectrum of the B-band is similar to the absorption curve of vertebrate rhodopsin. This was shown in our Euglena paper (James rt ul.. 1992) in a
06251
.0125-
01 400
450
500
Wavelength
550
600
650
(nm)
FIGURE 4. Chlumydomonus: eyespot spectra to show localization. Curve (1) 3 pm probe placed at center of eyespot Curve (2) Same probe moved I pm from center. Absolute O.D. shown.
granule.
EYESPOT
CHLAMYDOMONAS
550
500 Wavelength FIGURE 5. Chfamydomonas:
1597
600
650
(nm)
effect of varying probe size. Curve (1) 3 pm probe at eyespot center. Probe size increased to 4 pm [curve (2)], to 5 pm [curve (3)], to 6 pm [curve (4)]. 0.D.s in text.
comparison of the B-band with the spectral absorbance of three rhodopsins using the same instrument and techniques as in the eyespot analyses. Here we point out (Fig. 7) the same agreement of the B-band spectrum with the rhodopsin of the grass frog (Rana pipiens) whose maximum is at 502 nm. While the Chlamydomonas and Euglena eyespot spectra are similar in all the properties listed above, there appears to be one difference worthy of note. In the EugIena experiments, the A-band spectrum, while not
changing in magnitude with the 90” shift in polarization form, did undergo a red shift from about 414 to 432 nm (Table 1 in James ef al., 1992). At the same time the spectral maximum of the B-band remained fixed at about 494nm. In contrast, no such red shift was observed for the Chlamydomonas A-band. In 22 eyespots (44 recordings) the A-band was found to peak at 424 & 12.6 nm and 415 f 5.5 nm, respectively, in the parallel (axial) to perpendicular change in polarization plane. In the light of the many similarities in the spectral
80
g 60 aI .? cl E 40
0 400
450
500 Wavelength
550
600
650
(nm)
FIGURE 6. Chlamydomonus: effect of changing polarization. Curve (1) Probe polarized parallely (axially). Curve (2) Probe polarization perpendicular. Same probe, same eyespot. Curve (3) Repeat of perpendicular plane. Curve (4) Probe returned to parallel orientation. Normalized 0.D.s: 0.0844 [curve (l)], 0.0765 [curve (2)], 0.0857 [curve (3)], 0.0838 [curve (4)].
F. C‘RESC‘ITELLI
159x
TABLE Spectral
maximum
A-band
I. Spectral
properties
(nm)
of
1I eyespot>
O.D. maximum A-band
B-band
O.D. ratio
B-band
Polurization axis parallel to rhe anterior-posterior axis of cell 0.0439 + 0.0100 418.4 F 11.9 479.6 k 7.5 0.0627 f 0.0179
1.43
Polarization axis perpendicular IO anterior~-posterior axis ofcell 0.0362 + 0.0228 2.04 417.3 + 6.0 490.5 + 5.9 0.0737 + 0.0428 This table should
be compared
with Table
1 of the previous
paper.
curves of these two species this difference in behavior of the two A-bands is not immediately obvious. A possible explanation for this difference will be discussed in the next section in terms of the difference in structure of the stigma of these two species.
INTERPRETATIONS AND CONCLUSIONS
Our results are interpreted as positive answers to the two questions posed in the Introduction. First, we are able to relate the Chlumydomonas eyespot spectrum (B-band) to the expectations of the chemical findings and action spectra of Foster et al. (1984). A rhodopsin-type absorption curve for Chlamydomonas was predicted by these results and such a curve is here recorded (Fig. 7). This would appear to encircle the evidence for a retinalbased eyespot photopigment, which, since 1984, has grown to a convincing level. If an eyespot absorption differing from that of rhodopsin had been our finding this would have created problems in respect to the chemical results of the Foster group or else would have made suspect our microspectrophotometric probings. Fortunately, no such contradiction appeared and spectrophotometry has confirmed the claims of a rhodopsinlike system in Chlamydomonas.
YI ui.
The answer to the second question IS 111~11 thu nuture of the photopigment in E. gracilis, thus far hasod onI> on spectrophotometry. is better defined b> the findings that the Chlamydomonas eyespot spectrum is identical in form and properties to that of E. gruc’i/i.s. It IS logical LO assume that this similarity is based on a biochemical similarity. A common photochemistry may have e\~olveti in these two ancestral life forms. One difference in the eyespot spectra of these two species, the A-band red shift related to probe beam polarization in Euglena and its absence in (‘h/am!,domonas may be associated with the major difference in the structure of the stigma of these two species. If we assume, as we did for Euglena, that the Chlamydomonus B-band has its origin in a structure comparable to the Euglena PFB and the A-band in the stigma, the structure of the stigma then becomes relevant to the presence or absence of the A-band red shift. The eyespot of Chlam~~domonas differs from that of Euglenu in several ways. It is an intrachloroplastic organelle. there is no structure comparable to the PFB and the two emergent flagella are removed from immediate contact with the eyespot (Fig. 8). The Chlamydomonus stigma consists of stacked layers of pigmented granules alternating with unpigmented layers, the whole forming a reflective quarter wave assembly (Foster & Smyth, 1980). In contrast. the extrachloroplastic stigma of Euglena. examined by Walne and Arnott (1967) in E. granulata, consists of 5&60 granules of varying sizes, irregularly disposed: the whole forming a slightly curved body adjacent to the PFB. The Euglena stigma is neither layered, nor reflective. nor has it anisotropic character. How these differences in structure may be associated with the absence and presence of the A-band red shift in the two species is not immediately obvious. but a guess would be that light scattering for the two polarization forms of light
20-
0
I
450
’
’
’
’
I
500
’
Wavelength
550
600
(nm)
FIGURE 7. Comparison of Chlumydomonus B-spectrum [curve (l)] with a rhodopsin absorption [curve (2)] recorded with the same technique from the isolated retinal rod of a frog (Ram pipiens). Normalized 0.D.s: 0.0667 [curve (I)]. 0.0204 [curve (211.
CHLAMYDOMONAS
FIGURE 8. A sketch of Chlamydomonas to show the eyespot (E) with its granules over which is the thickened plasma membrane (darkened) which is believed to house the photopigment. The two emergent flagella (F) are not in contact with the eyespot. The eyespot is within a chloroplast (C). Other structures are the nucleus (N), the mitochondria (M), the pyrenoid (P). The bar indicates 1 pm. This figure to be compared to Fig. l(B) of preceding paper.
our probe beam could differ significantly and lead to different polarization effects in the two species which could, through absorption by optically active molecules, lead to spectral absorption shifts (optical rotatory dispersion effects). The effects of scattering in the layered structure of the stigma of Chlumydomonus could be minimal or quite different from scattering in the Euglena stigma, and this is suggested by the strikingly beautiful colored reflections that have been seen associated with such quarter-wave stigmas (Foster & Smyth, 1980). The site of origin of the B-band in Chlamydomonas is also a problem. We have assumed that the quasicrystalline PFB of E. gracifis (Piccinni & Mammi, 1978) houses the pigment responsible for the B-band, and its location close to the base of the flagellum is optimal for the transfer of excitation from the photopigment to the flagellum. Chlamydomonas does not have a PFB and its photopigment is assumed to be in the plasma membrane overlying the stigma (Foster et al., 1984). Walne and Arnott (1967) reported the plasma membrane over their granules to be thicker than normal and to protrude slightly, and the plasma membrane in this region appears to be structurally unique (Nultsch & Hader, 1988). The anisotropic B-band, assumed to arise from the PFB in Euglena has its source in a comparable structure in in
EYESPOT
1599
Chlamydomonas. This could be the overlying thickened membrane which houses the photopigment which, as in the vertebrate retina, is aligned with its preferred absorption in one plane. Differing from Euglena, the eyespot complex and flagellar base of Chlamydomonas are not in immediate proximity (Fig. 8) and this is beautifully demonstrated in a study by Harz and Hegemann (1991). These investigators employed the suction pipette technique in which the cell held on to the end of a 3 pm tip pipette was photically stimulated and the resulting current transients were recorded with the voltage clamp technology. Two clearly defined currents were found, one originating in the eyespot area, the second from the region of the flagellum removed from the eyespot. Significantly, the photoreceptor current from the eyespot gave a rhodopsin-like spectral response and was elicited with little delay, while the flagellar current showed all-or-none behavior with respect to the stimulus and was elicited within 1l-30 msec, depending on stimulus intensity. Some of the differences in structure between the eyespots of these two algae are pictured in Fig. 8 of this paper which is to be compared to Fig. 6 of the previous paper. The claim of a rhodopsin-like system in the eyespot of E. gracilis has to be weighed against the several proposals in the literature that a flavin is the eyespot photopigment (reviewed in James et al., 1992). Neither from Euglena nor Chlamydomonas have we recorded spectra that could be identified as flavins. Neither the A-B nor C-D curve of this paper can be identified with the absorption spectra of Wolken (1977) or of Schmidt, Galland, Senger and Furuya (1990). We also see no identity of our curves with the Eugfena action spectra of Diehn (1969). This failure to detect flavins could be the result of our using metabolically deficient chlorophyllfree cells, but this interpretation is refuted by our finding of an A-B-spectrum in green cells of the Z-strain of E. gracilis. In contrast to these failures to detect flavins is our confirmation of a rhodopsin spectrum in Chlamydomonas, where both action spectra and chemical reactions have established the presence of a rhodopsin (Foster et al., 1984). The basic similarity of the Euglena and Chlamydomonas eyespot spectra is consistent with our theoretical interpretation that a rhodopsin-like pigment is the photopigment in the paraflagellar body of E. gracilis. A similar conclusion was reached by Gualtieri, Barsanti and Passarelli (1989). Now what of the granules of both our algal species that did not disappear from view in the 470-560-470 flip-flop test and gave the red-shifted C-D-spectrum of Fig. 2? One possible role for the unidentified pigment of these granules is suggested by the study of Kondo, Johnson and Hastings (1991), which revealed the presence of a peak at about 520 nm in the action spectrum of circadian clock resetting for C. reinhardtii. The authors concluded that the pigment responsible (unidentified) differs from the rhodopsin-like system of phototaxis. Our broad D-band (Fig. 2) absorbs over the same wavelength as the 520-peak of Kondo et al. (1991) and suggests that the C-D granules could be the functional
1600
F. CRESCITELLl
elements in setting the circadian rhythm. In any case this comparison points out the possibility of separate photopigments responsible for the separate photoresponses of these algae.
REFERENCES Diehn, B. (1969). Action spectra of the phototactic responses in Euglena. Biochimica et Biophysics Acta, I77, 136-143. Foster, K. W. & Smyth, R. D. (1980). Light antennas in phototactic algae. Microbiological Reviews, 44, 572-630. Foster, K. W., Saranak, J., Patel, N., Zarilli, G., Okabe, M., Kline, T. & Nakanishi, K. (1984). A rhodopsin in the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas. Nature, 311, 756-759. Girard, J., Chua, N.-H., Bennoun, P., Schmidt, G. & Delosme, M. (1980). Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii. Current Genetics, 2, 215-221. Gualtieri, P., Barsanti, L. & Passarelli, V. (1989). Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis. Biochimica et Biophysics Acta, 993, 293-296. Harz, H. & Hegemann, P. (1991). Rhodopsin-regulated calcium currents in Chlamydomonas. Nature, 351, 489-491. James, T. W., Crescitelli, F., Loew, E. R. 8c McFarland, W. N. (1992). The eyespot of Euglena gracilis: Microspectrophotometry. Vision Research, 32, 1583-1591.
el LI/
Kondo. T., Johnson, C. H. & Hastmgs, J. W. (199 1). Action spectrum for resetting the circadian phototaxis rhythm in the CW 15 strain of Chlamydomonas (1991). Plan1 Physiology, 95. 197 205 Nultsch, W. & Hader, D.-P. (1988). Photomovement m motile mrcroorganisms-II. Photochemistry and Photobiolog_v. 47, 837 869. Olive, J.. Wollman, F.-A.. Bennoun, P. & Recouvreur. M. ( 1981). Ultrastructure of thylakoid membranes in Chlamydomonav rein hardtii: Evidence for variations in the partition coefficient of the light-harvesting complex-containing particles upon membrane fracture. Archioes of Biochemistry Biophysics, 208, 456 467 Piccinni, E. & Mammi, M. (1978). Motor apparatus of Euglenu gracilis: Ultrastructure of the basal portion of the flagellum and the paraflagellar body. Bollettino et Zoologia, 4.5, 405. 414. Schmidt, W., Galland, P., Senger, H. & Furuya, M. (1990). Microspectrophotometry of Euglena gracilis. Planta, 182, 375 381. Walne, P. L. & Arnott, H. J. (1967). The comparative ultrastructure and possible function of eyespots: Eugfena grarilis and Chlamydomonas eugametos. Planta, 77, 325-35 I. Wolken, J. J. (1977). Euglena: The photoreceptor system for phototaxis. Journal of Protozoology. 24. 518--522.
Acknowledgements-Aided by University of California Committee on Research grants to F.C. and T.W.J. and by grant EY-02178 from the National Institute of Health to F.C. and NSF grant DCB 9006550 to J.M.E. The microspectrophotometer was designed and built by an NIH grant to W.N.M., an Harch grant to E.R.L. and an ONR/DOD contract grant to E.R.L.
Vision Res. Vol. 32, NO. 9, pp. lS93-1600, 1992 Printed in Great Britain. All rights reserved
The Eyespot of Chlamydomonas reinhardtii: a Comparative Microspectrophotometric Study F. CRESCTTELLI,**
T. W. JAMES,* J. M. ERICKSON,*
E. R. LOEW,? W. N. McFARLANDS
Received 5 August 1991; in revisedform 4 MarCh 1992
The eyespot of C~l~y~~nus reinhardtii is believed to utilize a rhodopsin-like pigment in its responses to light. This paper examines its eyespot by means of microspectrophotometry with the finding of an absorption spectrum with two bands, an A-band in the blue, and a B-band in the green. This spectrmn is identical to that pre~ously recorded from the eyespot of Eugha grudis. AS with Eugienu the B-band was found to have dichroic character and its spectrum was similar to the absorption curve of rhodopsin. This A-B-spectrum was always recorded from a single granule in each cell. It is concluded that both E. gracifis and C. r&&r&ii may uti%zea rhodopsin-like pigment as the photopigment associated with the eyespot response to light. In both these algae a few particles in each cell were found whose spectra consisted of two other bands, C and D, blue- and red-shifted, respectively, relative to the eyespot A-B-bands. There is some reason to believe that the C-D-granules may also be involved in certain ~ght~on~olled activities of the cells. ChIamydomonas Eyespot
Rhodopsin
Spectral absorption
INTRODUCTION In the preceding paper (James, Crescitelli, Loew & McFarland, 1992) the spectral absorbance of individual eyespots of EuglePla gracilis was shown to consist of two absorption bands? one in the blue (A-band), the second in the green (B-band). Unlike the A-band, the B-band displayed dichroic character, a property that suggested the absorption of a pigment oriented in the anisotropic, quasi-crystalline paraflagellar body (PFB). The isotropic A-band was tentatively interpreted to be the absorption of a pigment in the orange-red stigma in close association with the PFB. A significant feature of this study was the similarity of the B-band to the spectral absorption curve of rhodopsin. Because of this finding attempts were made to obtain chemical evidence of a rhodopsinlike system in the eyespot but these were unsuccessful because photic bleaching did not occur so that the possible effects of hydroxylamine and of added retinal could not be examined. This idea of a rhodopsin-like photosystem in phototactic algae is not new because recent studies have reported chemical evidence that the eyespot of Chlamydomonas rein~~rdti~ does indeed possess such a photopigment. Taking advantage of mutant FN68 which is defective in the synthesis of retinal, Foster, Saranak, *Department of Biology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024-1606, U.S.A. tPhysiology Division of Biological Sciences and NYS College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. ICatalina Marine Science Center, University of Southern California, P.O. Box 396, Avalon, CA 90704, U.S.A. *Deceased.
Patel, Zarilli, Okabe, Kline and Nakanishi (1984) were able to demonstrate an increase in phototactic sensitivity by the addition of retinal and the resulting action spectrum was similar to the spectral absorption of bovine rhodopsin. Apparently, opsin was present in this mutant so that the addition of retinal restored the photopigment required for the light response. Along with our study of the Euglena eyespot (James et al., 1992) the evidence of a rhodopsin-like system in Chlamydomonas offers the possibility of advancing the subject in two directions by finding answers to two related questions: (a) what is the spectral absorbance of the Chlamydomonas eyespot and does it support the published chemical evidence of a retinal-based photopigment; and (b) what is the form of the Chlumydomonas eyespot spectrum? Is it like that of E. gracilis, with Aand B-bands, the latter band alone showing a dichroic property? If the answer to this second question .is positive it adds confidence to the hypothesis of a rhodopsin-like pigment in E. gracilis for which the necessary chemical evidence is lacking. A positive answer to both these questions would serve to further generalize the concept of an early phylogenetic origin of retinalbased pigments in biological photosensitivity. In this paper, therefore, we intend to present evidence that bears on both these questions. MATERIALS AND METHODS As already indicated (James et al., 1992), algal cells with chlorophyll offer complications in the recording of spectral absorbance in that the two main chlorophyll bands dominate the spectrum and make it difficult to
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sort out the eyespot response. For this reason Euglena cells were used that were bleached free of chlorophyll with streptomycin or by growth in the dark. Cells from these cultures yielded eyespot absorption curves free of chlorophyll contamination and provided data that were summarized in the Introduction. We also recorded the eyespot absorption of green cells of the Z-strain of E. gracilis and obtained the same A-B-spectrum, the Bband with similar dichroic behavior. This result was useful in showing that the A-B-spectrum is not the consequence of an impaired metabolism due to the deficiency of chlorophyll. For the present study, we used a chlorophyll-deficient mutant of C. reinhardtii, BF4/Ml8, initially obtained from J. Girard-Bascou and P. Bennoun. BF4/M18 is a double, nuclear mutant deficient in the light-harvesting chlorophyll complex associated with photosystem II (Olive et al., 1981) and lacking photosystem I (Girard et al., 1980). The spectral recordings were obtained with the same microspectrophotometer and procedures as in the Euglena study, already cited. The living cells, lightly compressed between two cover slips in a metal frame holder, were placed on the microscope stage and visualized on a video monitor which received the output of an infrared camera mounted on the exit optics of the microscope. A quiescent cell was selected, its eyespot was found with the field illuminated with light at 470 nm as in the Euglena study. On the monitor screen the Chlumy domonas eyespot was seen as a dark body on a lighter granular cytoplasmic background. Since bodies other than the eyespot were normally present, and their morphology was often not unlike that of the eyespot, it was necessary to distinguish these as elements different from the single eyespot in each cell. This was carried out by taking advantage of the eyespot color. The field illumination was set at 470 nm, changed to 560 nm and then
back to 470 nm, a flip-flop procedure that caused the eyespot to disappear from view and then to reappear. Granules other than the eyespot remained in view during this procedure. This technique did indeed pick out the eyespot. Only one granule in each cell responded positively to this 470-560-470 maneuver, and it was this granule, and not the others, from which the A--B spectrum to be described was obtained. Once the eyespot was located, the field was returned to infrared viewing and the light probe beam at 750 nm was turned on and adjusted to an appropriate dimension to fit the eyespot, the smallest being a square with 3 pm sides. The probe was then placed in the clear medium outside the cell and a spectral scan was made over the chosen wavelength span. The data were stored in the computer and the probe next positioned as close to the center of the selected eyespot as possible. A similar spectral scan was again made and the difference spectrum resulting from these two scans was viewed and then stored for later printing and study. The cells of Chlaml,domonas, though relatively small compared to those of Euglena, remained alive and motile for hours between the cover slips and we were able to collect some 300 recordings from about 200 different eyespots. The microspectrophotometer (MSP) was described in the previous paper and employed as there described. It was there described as an instrument capable of recording the correct spectra of a human red blood cell, a visual rod from the frog retina and of a didymium filter.
RESULTS It was quickly discovered that the granules that reacted to the flip-flop test yielded spectra characteristically different from those recorded from the granules that did not flip-flop. The former gave spectra (Fig. 1)
Wavelength FIGURE
1. Eyespot
spectra
showing
Y[ (I!
(nm)
A- and B-bands from E. gracilis (1) and C. reinhardtii are 0.0457 [curve (l)] and 0.0764 [curve (2)].
(2). The 100% normalized
0.D.s
CHLAMYDOMONAS
400
500
450 Wavelength
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EYESPO-I
550
600
(nm)
FIGURE 2. Spectral absorption of the two types of granules recorded from the C. reinhardtii eyespot. Curve (1) Granule that went out in 470-560 nm shift, showing A- and B-bands. Curve (2) Granule that remained in 47&560 nm shift, showing Cand D-bands. Granule 1 was an eyespot; granule 2 was next to eyespot. Normalized 0.D.s: 0.0871 [curve (l)], 0.0097 [curve (2)].
the A- and B-bands already known from the Euglena experiments, while the latter granules lacked these two bands, but not all were spectroscopically silent, for we consistently recorded from a few a double-banded spectrum consisting of what we call C- and D-bands shifted significantly from the locations of the A- and B-bands. As seen in Fig. 2, the C-band is blue-shifted by some 18-20 nm from the A-band, while the D-band is red-shifted by an even greater amount (33-35 nm) from the B-band. In addition, the form of the B- and
with
D-spectra differ in that the former is featureless while the latter is broader and seems to have a multi-banded structure (Fig. 2). The different spectral locations of the B- and D-bands explains why the eyespot disappeared in the flip from 470 to 560 nm while the granules responsible for the D-band persisted. Incidentally, after the discovery in Chlamydomonas of spots with C- and D-bands, we returned to E. gracilis and found similar bodies that remained in view in the 470-560-test and gave a spectrum with C- and D-bands (Fig. 3).
20-
0
FIGURE
“,“,,,,‘,,,,,,,,,,,,, 400
450 Wavelength
500 (nm)
550
600
3. The C- and D-bands from Euglena [curve (l)] and Chfumydomonm [curve (2)] from granules that remained in the 470-560-test. Normalized 0.D.s: 0.0435 [curve (l)], 0.0097 [curve (2)].
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As in E. gracilis, the Chlamydomonas A-B spectrum was found in a small and highly localized spot, and movement of the probe away from this spot by as little as 1 ,~rn resulted in a reduction or disappearance of the spectrum, an example being that of Fig. 4 where a 3 ,um probe was first placed at a point of largest response [curve (l)] and then moved 1 pm away from this point [curve (2)]. This rather precise localization favors the idea of a pigment in a very small region of the cell as the source of the A-B-spectrum. It is important to point out that it was never possible to obtain records with the A-band alone or the B-band alone, a situation that we also encountered in our Euglena work. This does not necessarily disprove the idea that the A- and B-bands originate in different structures within the eyespot complex, since over dimensions of the order of 3 pm and more, light scattering probably prevents the precise localization of probe beam to only one of the two possible structures. Another resemblance to the Euglena spectrum was the effect of varying probe size. In the E. gracilis eyespot a small probe centered on the active region gave a spectrum in which the A- and B-bands were fused into a broadly-rounded maximum. Increasing probe size caused a decrease of spectrum magnitude and brought out the A- and B-bands. A similar response was found in Chlamydomonas (Fig. 5) where a probe with 3.0 pm sides yielded a spectrum [curve (I)] in which the two bands were not clearly distinguished. Increasing probe size to 4 pm [curve (2)], to 5 pm [curve (3)] and to 6 pm [curve (4)] led to a progressive separation of the two bands. In addition, the maximum (100%) O.D. values of these four recordings decreased progressively (0.1160, 0.0805, 0.0469, 0.0239). These two effects of probe beam size may be ascribed to a dilution of pigment concentration as the probe size increased to include
er ui
cytoplasmic areas outside the eyespot that iackcd pugment, although a possible effect of light scattering C;~IF not be discounted. Perhaps the most significant property of the c Mmzr domonas spectrum, as with E. gracilis, was the effect observed by a 90” change in polarization of the probe from a parallel to a perpendicular orientation with respect to the cell anterior- posterior axis. Compared to Euglena, the Chlamydomonas cell is smaller and rounder, so that determining the cell axis was difficult and uncertain. We therefore employed the location of the Ragellar swirl as indicative of the anterior pole of the cell. The result of this 90“ change in polarization axis was II selective reduction of the B-band. suggesting an anisotropic structure as the source of this absorption (Fig. 6). Figure 6, selected because of its relatively high dichroic ratio (2.2) is at the upper level of ratios obtained from eyespots, which show a considerable variance. In one series of recordings using the same eyespot and IO successive probings with the same 3.0pm probe, dichroic ratios from 1.30 to 1.92 were obtained. The variability was even greater when a comparison of different eyespots was made. This variability is perhaps to be expected when recording from living cells in which the eyespot preferred axis is changing constantly due to cytoplasmic movements. In any case, the result pictured in Fig. 6 along with the statistical data of Table 1 confirms the idea of an anisotropic structure as the source of the B-band in Chlamydomonas. As with Euglena no evidence for anisotropy was seen for the A-band (Table 1). From a biological point of view perhaps the most interesting similarity in the results with these two algal species is that the spectrum of the B-band is similar to the absorption curve of vertebrate rhodopsin. This was shown in our Euglena paper (James rt ul.. 1992) in a
06251
.0125-
01 400
450
500
Wavelength
550
600
650
(nm)
FIGURE 4. Chlumydomonus: eyespot spectra to show localization. Curve (1) 3 pm probe placed at center of eyespot Curve (2) Same probe moved I pm from center. Absolute O.D. shown.
granule.
EYESPOT
CHLAMYDOMONAS
550
500 Wavelength FIGURE 5. Chfamydomonas:
1597
600
650
(nm)
effect of varying probe size. Curve (1) 3 pm probe at eyespot center. Probe size increased to 4 pm [curve (2)], to 5 pm [curve (3)], to 6 pm [curve (4)]. 0.D.s in text.
comparison of the B-band with the spectral absorbance of three rhodopsins using the same instrument and techniques as in the eyespot analyses. Here we point out (Fig. 7) the same agreement of the B-band spectrum with the rhodopsin of the grass frog (Rana pipiens) whose maximum is at 502 nm. While the Chlamydomonas and Euglena eyespot spectra are similar in all the properties listed above, there appears to be one difference worthy of note. In the EugIena experiments, the A-band spectrum, while not
changing in magnitude with the 90” shift in polarization form, did undergo a red shift from about 414 to 432 nm (Table 1 in James ef al., 1992). At the same time the spectral maximum of the B-band remained fixed at about 494nm. In contrast, no such red shift was observed for the Chlamydomonas A-band. In 22 eyespots (44 recordings) the A-band was found to peak at 424 & 12.6 nm and 415 f 5.5 nm, respectively, in the parallel (axial) to perpendicular change in polarization plane. In the light of the many similarities in the spectral
80
g 60 aI .? cl E 40
0 400
450
500 Wavelength
550
600
650
(nm)
FIGURE 6. Chlamydomonus: effect of changing polarization. Curve (1) Probe polarized parallely (axially). Curve (2) Probe polarization perpendicular. Same probe, same eyespot. Curve (3) Repeat of perpendicular plane. Curve (4) Probe returned to parallel orientation. Normalized 0.D.s: 0.0844 [curve (l)], 0.0765 [curve (2)], 0.0857 [curve (3)], 0.0838 [curve (4)].
F. C‘RESC‘ITELLI
159x
TABLE Spectral
maximum
A-band
I. Spectral
properties
(nm)
of
1I eyespot>
O.D. maximum A-band
B-band
O.D. ratio
B-band
Polurization axis parallel to rhe anterior-posterior axis of cell 0.0439 + 0.0100 418.4 F 11.9 479.6 k 7.5 0.0627 f 0.0179
1.43
Polarization axis perpendicular IO anterior~-posterior axis ofcell 0.0362 + 0.0228 2.04 417.3 + 6.0 490.5 + 5.9 0.0737 + 0.0428 This table should
be compared
with Table
1 of the previous
paper.
curves of these two species this difference in behavior of the two A-bands is not immediately obvious. A possible explanation for this difference will be discussed in the next section in terms of the difference in structure of the stigma of these two species.
INTERPRETATIONS AND CONCLUSIONS
Our results are interpreted as positive answers to the two questions posed in the Introduction. First, we are able to relate the Chlumydomonas eyespot spectrum (B-band) to the expectations of the chemical findings and action spectra of Foster et al. (1984). A rhodopsin-type absorption curve for Chlamydomonas was predicted by these results and such a curve is here recorded (Fig. 7). This would appear to encircle the evidence for a retinalbased eyespot photopigment, which, since 1984, has grown to a convincing level. If an eyespot absorption differing from that of rhodopsin had been our finding this would have created problems in respect to the chemical results of the Foster group or else would have made suspect our microspectrophotometric probings. Fortunately, no such contradiction appeared and spectrophotometry has confirmed the claims of a rhodopsinlike system in Chlamydomonas.
YI ui.
The answer to the second question IS 111~11 thu nuture of the photopigment in E. gracilis, thus far hasod onI> on spectrophotometry. is better defined b> the findings that the Chlamydomonas eyespot spectrum is identical in form and properties to that of E. gruc’i/i.s. It IS logical LO assume that this similarity is based on a biochemical similarity. A common photochemistry may have e\~olveti in these two ancestral life forms. One difference in the eyespot spectra of these two species, the A-band red shift related to probe beam polarization in Euglena and its absence in (‘h/am!,domonas may be associated with the major difference in the structure of the stigma of these two species. If we assume, as we did for Euglena, that the Chlamydomonus B-band has its origin in a structure comparable to the Euglena PFB and the A-band in the stigma, the structure of the stigma then becomes relevant to the presence or absence of the A-band red shift. The eyespot of Chlam~~domonas differs from that of Euglenu in several ways. It is an intrachloroplastic organelle. there is no structure comparable to the PFB and the two emergent flagella are removed from immediate contact with the eyespot (Fig. 8). The Chlamydomonus stigma consists of stacked layers of pigmented granules alternating with unpigmented layers, the whole forming a reflective quarter wave assembly (Foster & Smyth, 1980). In contrast. the extrachloroplastic stigma of Euglena. examined by Walne and Arnott (1967) in E. granulata, consists of 5&60 granules of varying sizes, irregularly disposed: the whole forming a slightly curved body adjacent to the PFB. The Euglena stigma is neither layered, nor reflective. nor has it anisotropic character. How these differences in structure may be associated with the absence and presence of the A-band red shift in the two species is not immediately obvious. but a guess would be that light scattering for the two polarization forms of light
20-
0
I
450
’
’
’
’
I
500
’
Wavelength
550
600
(nm)
FIGURE 7. Comparison of Chlumydomonus B-spectrum [curve (l)] with a rhodopsin absorption [curve (2)] recorded with the same technique from the isolated retinal rod of a frog (Ram pipiens). Normalized 0.D.s: 0.0667 [curve (I)]. 0.0204 [curve (211.
CHLAMYDOMONAS
FIGURE 8. A sketch of Chlamydomonas to show the eyespot (E) with its granules over which is the thickened plasma membrane (darkened) which is believed to house the photopigment. The two emergent flagella (F) are not in contact with the eyespot. The eyespot is within a chloroplast (C). Other structures are the nucleus (N), the mitochondria (M), the pyrenoid (P). The bar indicates 1 pm. This figure to be compared to Fig. l(B) of preceding paper.
our probe beam could differ significantly and lead to different polarization effects in the two species which could, through absorption by optically active molecules, lead to spectral absorption shifts (optical rotatory dispersion effects). The effects of scattering in the layered structure of the stigma of Chlumydomonus could be minimal or quite different from scattering in the Euglena stigma, and this is suggested by the strikingly beautiful colored reflections that have been seen associated with such quarter-wave stigmas (Foster & Smyth, 1980). The site of origin of the B-band in Chlamydomonas is also a problem. We have assumed that the quasicrystalline PFB of E. gracifis (Piccinni & Mammi, 1978) houses the pigment responsible for the B-band, and its location close to the base of the flagellum is optimal for the transfer of excitation from the photopigment to the flagellum. Chlamydomonas does not have a PFB and its photopigment is assumed to be in the plasma membrane overlying the stigma (Foster et al., 1984). Walne and Arnott (1967) reported the plasma membrane over their granules to be thicker than normal and to protrude slightly, and the plasma membrane in this region appears to be structurally unique (Nultsch & Hader, 1988). The anisotropic B-band, assumed to arise from the PFB in Euglena has its source in a comparable structure in in
EYESPOT
1599
Chlamydomonas. This could be the overlying thickened membrane which houses the photopigment which, as in the vertebrate retina, is aligned with its preferred absorption in one plane. Differing from Euglena, the eyespot complex and flagellar base of Chlamydomonas are not in immediate proximity (Fig. 8) and this is beautifully demonstrated in a study by Harz and Hegemann (1991). These investigators employed the suction pipette technique in which the cell held on to the end of a 3 pm tip pipette was photically stimulated and the resulting current transients were recorded with the voltage clamp technology. Two clearly defined currents were found, one originating in the eyespot area, the second from the region of the flagellum removed from the eyespot. Significantly, the photoreceptor current from the eyespot gave a rhodopsin-like spectral response and was elicited with little delay, while the flagellar current showed all-or-none behavior with respect to the stimulus and was elicited within 1l-30 msec, depending on stimulus intensity. Some of the differences in structure between the eyespots of these two algae are pictured in Fig. 8 of this paper which is to be compared to Fig. 6 of the previous paper. The claim of a rhodopsin-like system in the eyespot of E. gracilis has to be weighed against the several proposals in the literature that a flavin is the eyespot photopigment (reviewed in James et al., 1992). Neither from Euglena nor Chlamydomonas have we recorded spectra that could be identified as flavins. Neither the A-B nor C-D curve of this paper can be identified with the absorption spectra of Wolken (1977) or of Schmidt, Galland, Senger and Furuya (1990). We also see no identity of our curves with the Eugfena action spectra of Diehn (1969). This failure to detect flavins could be the result of our using metabolically deficient chlorophyllfree cells, but this interpretation is refuted by our finding of an A-B-spectrum in green cells of the Z-strain of E. gracilis. In contrast to these failures to detect flavins is our confirmation of a rhodopsin spectrum in Chlamydomonas, where both action spectra and chemical reactions have established the presence of a rhodopsin (Foster et al., 1984). The basic similarity of the Euglena and Chlamydomonas eyespot spectra is consistent with our theoretical interpretation that a rhodopsin-like pigment is the photopigment in the paraflagellar body of E. gracilis. A similar conclusion was reached by Gualtieri, Barsanti and Passarelli (1989). Now what of the granules of both our algal species that did not disappear from view in the 470-560-470 flip-flop test and gave the red-shifted C-D-spectrum of Fig. 2? One possible role for the unidentified pigment of these granules is suggested by the study of Kondo, Johnson and Hastings (1991), which revealed the presence of a peak at about 520 nm in the action spectrum of circadian clock resetting for C. reinhardtii. The authors concluded that the pigment responsible (unidentified) differs from the rhodopsin-like system of phototaxis. Our broad D-band (Fig. 2) absorbs over the same wavelength as the 520-peak of Kondo et al. (1991) and suggests that the C-D granules could be the functional
1600
F. CRESCITELLl
elements in setting the circadian rhythm. In any case this comparison points out the possibility of separate photopigments responsible for the separate photoresponses of these algae.
REFERENCES Diehn, B. (1969). Action spectra of the phototactic responses in Euglena. Biochimica et Biophysics Acta, I77, 136-143. Foster, K. W. & Smyth, R. D. (1980). Light antennas in phototactic algae. Microbiological Reviews, 44, 572-630. Foster, K. W., Saranak, J., Patel, N., Zarilli, G., Okabe, M., Kline, T. & Nakanishi, K. (1984). A rhodopsin in the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas. Nature, 311, 756-759. Girard, J., Chua, N.-H., Bennoun, P., Schmidt, G. & Delosme, M. (1980). Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii. Current Genetics, 2, 215-221. Gualtieri, P., Barsanti, L. & Passarelli, V. (1989). Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis. Biochimica et Biophysics Acta, 993, 293-296. Harz, H. & Hegemann, P. (1991). Rhodopsin-regulated calcium currents in Chlamydomonas. Nature, 351, 489-491. James, T. W., Crescitelli, F., Loew, E. R. 8c McFarland, W. N. (1992). The eyespot of Euglena gracilis: Microspectrophotometry. Vision Research, 32, 1583-1591.
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Kondo. T., Johnson, C. H. & Hastmgs, J. W. (199 1). Action spectrum for resetting the circadian phototaxis rhythm in the CW 15 strain of Chlamydomonas (1991). Plan1 Physiology, 95. 197 205 Nultsch, W. & Hader, D.-P. (1988). Photomovement m motile mrcroorganisms-II. Photochemistry and Photobiolog_v. 47, 837 869. Olive, J.. Wollman, F.-A.. Bennoun, P. & Recouvreur. M. ( 1981). Ultrastructure of thylakoid membranes in Chlamydomonav rein hardtii: Evidence for variations in the partition coefficient of the light-harvesting complex-containing particles upon membrane fracture. Archioes of Biochemistry Biophysics, 208, 456 467 Piccinni, E. & Mammi, M. (1978). Motor apparatus of Euglenu gracilis: Ultrastructure of the basal portion of the flagellum and the paraflagellar body. Bollettino et Zoologia, 4.5, 405. 414. Schmidt, W., Galland, P., Senger, H. & Furuya, M. (1990). Microspectrophotometry of Euglena gracilis. Planta, 182, 375 381. Walne, P. L. & Arnott, H. J. (1967). The comparative ultrastructure and possible function of eyespots: Eugfena grarilis and Chlamydomonas eugametos. Planta, 77, 325-35 I. Wolken, J. J. (1977). Euglena: The photoreceptor system for phototaxis. Journal of Protozoology. 24. 518--522.
Acknowledgements-Aided by University of California Committee on Research grants to F.C. and T.W.J. and by grant EY-02178 from the National Institute of Health to F.C. and NSF grant DCB 9006550 to J.M.E. The microspectrophotometer was designed and built by an NIH grant to W.N.M., an Harch grant to E.R.L. and an ONR/DOD contract grant to E.R.L.
