Photochemistry and Phutuhrolugy, 1975. Vol. 21, pp. 351 354.

Pergamon Press. Printed i n Great Britain

PHOTOTACTIC RESPONSE OF e H u M Y m M o N A s TO FLASHES OF LIGHT-I. RESPONSE OF CELL POPULATIONS MARYELLAFEINLEIB Biology Department, Tufts University, Medford, Massachusetts 02155 U.S.A. (Received 26 August 1974;accepted 16 January 1975) Abstract-Chlamydomonas reinhardi responds phototactically to a single, very short flash of blue light ( 6 4 p).Net oriented response of a cell population is monitored photometrically, using the “population system” of Feinleib and Curry (1967). A single high-intensity flash elicits a small, but definite net movement away from the stimulus source. Repetitive flashing at low frequency (between 8 and 60 flashes per min) and at the same intensity elicits a prolonged response in the same direction. Net phototactic response to single or repetitive flashes varies with stimulus intensity in the same way as does response to continuous light (Feinleib and Curry, 1971b); response is positive at low intensity and negative at high intensity. These data indicate that at least some cells become oriented in responsc to a short flash. The occurrence of such a response has implications for the mechanism of phototactic orientation. If almost all the cells responded, one would assume that Chlamydomonas perceives light direction instantaneously by detecting an absorption gradient within the cell. Unequivocal interpretation of the short-flash response requires examination of the behavior of individual cells.

INTRODUCTION

Flagellated algae such as Chlamydomonas and Euglena can swim directly toward (or away from) a light source (topotaxis; Pfeffer, 1904). Since this response is oriented with respect to stimulus direction, the organism must have some mechanism for detecting that direction; in most species this mechanism is not yet understood. There are two basic ways in which light direction may be detected (Haupt, 1966; Hand and Davenport, 1970; Nultsch, 1970; Feinleib and Curry, 1971a). The organism may perceive light direction by comparing the light absorbed in two regions of the cell at one instant in time (e.g., the left and right halves of a photoreceptor organelle). For convenience, this will be referred to as the “one-instant” mechanism, since the cell can obtain complete information on light direction instantaneously, without changing position with respect to the light source. Alternatively, the organism may detect light direction by a ‘two-instant’ mechanism; i.e. by comparing the light absorbed in one region of the cell at ‘two instants’ in time. With a continuous stimulus, the cell can obtain these two absorption ‘readings’ by taking two different positions with respect to the light source. The photoreceptor organelle will absorb different amounts of light in the two positions if, e.g. it is asymmetrically shaped or if it is asymmetrically located with respect to other light-absorbing components of the cell. It is commonly held that Euglena detects light direction by a ‘two-instant’ mechanism (Mast, 1911; Diehn, 1969, 1973; Nultsch, 1970). According to this hypothesis, the photoreceptor resides in a swelling at the flagellar base (paraflageller body) and the nearby

stigma acts as a shading device. In unilateral light, the stigma periodically shades the paraflagellar body as the cell rotates on its longiiudinal axis. In the case of positive phototaxis, the resulting decrease in intensity on the photoreceptor causes the cell to turn in a morphologically-defined direction; namely, toward the ‘dorsal’ or stigma-containing side. Once the organism is swimming directly toward the light source, the periodic shading stops. Another version of the ‘two-instant’ mechanism has been proposed for the dinoflagellate Gyrodiriium (Schmidt and Hand, personal communication). When the organism is exposed to a stimulus beam normal to its swimming direction, it continues to swim forward and to rotate on its longitudinal axis until the sulcus-furrow junction faces the stimulus source. At that point, the cell stops all motion for one to five seconds. It then turns directly toward the side bearing the sulcus-furrow junction; i t . toward the stimulus source, and begins swimming toward the light. Schmidt and Hand suggest that the photoreceptor in Gyodinium is located at the sulcus-furrow junction. When the photoreceptor becomes directed toward the light, and only then, the cell experiences a sudden increase in intensity and turns toward the light source. There is no dear evidence to date that any uniceltular flagellate detects light direction by a ‘one-instant’ mechanism, although this kind of orientation apparently operates in some filamentous blue-green algae (Nultsch, 1970). The present study was designed to determine whether Chlamydomonas becomes oriented by a one or by a ‘two-instant’ mechanism. The approach is to expose the cells to a single very short flash of light in the microsecond range. If almost all

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the cells become oriented to such a short flash, it would seem likely that they can detect light direction instantaneously, without changing position.

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MATERIALS AND METHODS

Oriented phototactic response of a cell population is measured using the ‘population system’ of Feinleib and Curry (1967). The net movement of algae toward (or away from) a stimulus is monitored with dim red light and is recorded continuously as a difference in output between two photocells. This recorded signal is referred to as a net-response curve; the slope of this curve is taken as the index of net response. The stimulus-light source for these experiments is a stroboscopic lamp (General Radio Strobotac Type 153I A ) with a flash duration of 6 . 4 at ~ one-third ~ maximum intensity. The flashes are triggered manually via a switch connected to the lamp. The light is passed through a water filter and a broad band-pass blue filter (Corning 4-96). Light intensity is regulated with Bausch & Lomb neutraldensity filters. Intensities are measured with an Eppley bismuth-silver thermopile, calibrated by Eppley against a standard lamp. Duration and relative intensity of the stroboscopic flash are determined using an EG & G photodiode. with a rise time of less than 10 ns; the output is displayed on an oscilloscope. Chlamydornorias reinhardi, wild-type + (No. 137c) is used for all experiments. The cells are grown aseptically in 200m6P aliquots of liquid ‘minimal’ medium (Levine and Ebersold, 1958) with constant aeration. Cultures are started from inocula of about 5 x lo6 cells and are maintained in constant light of 6.1 x lo3 lux (‘cool-white’ fluorescent) at 24 5 2°C. Cultures are used at about 72 h following inoculation. Unless noted otherwise, a fresh sample of algae is used for each test exposure. The sample is adjusted to a standard optical density at 750 nm, using medium filtered from an identical culture. The algal sample is then transferred to the test chamber and is exposed for 1 rnin to the red monitoring light alone before introduction of the stimulus. RESULTS AND DISCUSSION

A single 6.4 ps flash of the stroboscopic lamp elicits a small but definite net response in the population system. Some typical net-response curves recorded in

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Figure I . Net response of a cell population to single highintensity lldshes ol’ blue light (200 pW-cm’). These curves were traced from recordings of output photocell 2 minus output photocell 1 us time, obtained in the population system. (A) Response to a single 6.4 p s flash, presented at time 0 and again at time 2 rnin. (B) Response to repetitive llashing at a frequency of 8 flashes per min. (C) Response to repetitive flashing at 60 flashes per min.

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Figure 2. Net response of a cell population to single flashes of blue light at high intensity (A) and at low intensity (B). Recordings obtained as in Fig. I. (A) Response at 200 pW-cm-’, to a single flash and to repetitive flashing at 60 flashes per min. (B) Response at 12.5 pW. to a single flash and to repetitive flashing at 60 flashes per min.

these experiments are shown in Fig. I. Net response (output photocell 2 minus output photocell I ) is plotted against time. The base line, obtained in red light only, indicates no difference in output. A single high-intensity flash occurs at time 0 (Fig. I , part A). This stimulus elicits a small net negative response, lasting between 10 and I5 s. A second flash is presented to the algal sample at time 2 min. with the same result. These small responses become more convincing when it is found that repetitive flashing at low frequency elicits a prolonged response in the same direction. In Fig. IB, eight flashes are given during a I-min interval at the same high intensity. In this case, net response is distinctly negative, presuma hly representing a summation of single-flash responses. Fig. IC shows the net response to repetitive flashing at 60 flashes per min (1 per s). At the end of 1 min of stimulation, a distinct accumulation of cells can be seen at the far wall of the swimming chamber. The net phototactic response to single flashes varies with stimulus intensity in the same way as does the response to continuous light (Feinleib and Curry, 1971b). The first pair of recordings in Fig. 2(A) shows the net response of an algal sample to high-intensity flash stimulation. Net response is negative to the single flash as well as to repetitive flashing at 60 per min. In the second pair of recordings (B), the stimulus intensity has been lowered by a factor of 16. Here the net response is positive; it is very small following the single flash, but perfectly clear with repetitive flashing. In Fig. 3, the slope of the net response curve is plotted as a function of stimulus intensity for single flashes and for repetitive flashes at 60 per min. The single-flash responses are so small that the slope measurements are inaccurate; nonetheless. the curve for single flashes agrees roughly in shape with that for repetitive flashes. Both curves resemble the one published earlier for net response to continuous light (Fig. 4 in Feinleib and Curry, 1971b). These data sug-

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Phototactic response of Chlarnydomonas

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Figure 3. The relation between net response of a cell population and stimulus intensity for single flashes and for repetitive stimulation at 60 flashes per min. Intensity is plotted on a logarithmic scale. The lines drawn through the points indicate the range of values.

gest that at least some of the organisms in the chamber become oriented following a single, very short flash of light. Movement in response to a short light flash has also been recorded in Euglena (Diehn, 1969), using a 'phototaxigraph' (modified after Lindes et al., 1965). Diehn illuminated a region of a Euglena suspension with white light for 1 ms. Cells subsequently accumulated in the region which had been illuminated. Diehn concludes that this phenomenon does not represent phototactic orientation. He explains it instead as the indirect result of a shock reaction which momentarily prevents Euglena from leaving the illuminated zone. Meanwhile, cells outside that zone continue normal movement. The result is a net influx of cells into the region, accounting for the recorded response. O n the basis of the phototaxigraph data alone, one cannot dismiss the possibility that cells outside the illuminated zone show a n oriented response to light reflected from organisms inside the zone. However, Diehn's observations through the microscope (1 969, 1972, 1973) are consistent with his interpretation of the phototaxigraph results. We have considered the possibility that the apparent net response to a single flash recorded in our population system may merely be the indirect result of a shock or stop response. For example, if most of the cells in the front half of the chamber (nearer to the light source) stopped in response to the flash. while most of the cells in the rear half continued to move, the result would be a net movement of cells

toward the light. It would be difficult, however, to devise a consistent hypothesis along these lines that accounts both for positive phototaxis a t low intensity and for negative phototaxis at high intensity. We thus consider it most likely that, in Chlamydomonas, some phototactic orientation occurs in response to a flash. In order to interpret the short-flash response unequivocally, we must examine the behavior of individual cells. If directed response to a flash indeed occurs, the next step is to establish the percentage of cells which respond. If almost all the cells become oriented, they presumably detect light direction by a 'one-instant' mechanism; if only a fraction of the population responds, then a 'two-instant' mechanism may be operating, as in Gyrodinium (Schmidt and Hand, personal communication). In the latter case, the only cells to become oriented might be. those in which the photoreceptor is directed toward the light source at the instant of the flash. The response of individual cells is currently being studied in this laboratory using a video-microscope system (Boscov, 1974). Ackriowledgements-Supported by a grant from the National Science Foundation (GB-34309). The author is indebted to Dr. R. L. Carpenter and to Dr. J. Feinleib for their assistance in calibrating the stroboscopic lightsource, to Mr. J. s. Boscov for his help with intensity measurements, and to Dr. R . P. Levine for supplying the stock cultures of Chlamydomonas.Special thanks go to Dr. G. M. Curry for his critical discussions of the experiments and the manuscript.

REFERENCES

Boscov, J. S. (1974) M.S. thesis., Tufts University. Medford, Mass. Diehn, B. (1969) E x p . Cell Rrs. 56, 375-381. Diehn, B. (1972) In The Behaoior of Microorgatiiswu (Edited by J. Adler), pp. 83-90. Plenum, New York. Diehn, B. (1973) Science 181. 1009-1015. Feinleib, M. E. and G . M. Curry (1967) Physiol. Plantarurn 20, 1083-1095.

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MARYELLAFEINLEIB Feinleib, M. E. and G . M . Curry (1971a) In Handbook of Sensory Physiology, Principles of Receptor Physiology (Edited by W. R. Loewenstein), Vol. I., pp. 366395. Springer, Berlin. Feinleib, M. E. and G . M. Curry (1971b) Physiol. Plantarum 25. 346-352. Hand, W. G. and D. Davenport (1970) In -Photobiology of Microorganisms (Edited by P. Halldal), V V . 253-282. Haipt. W. (1959) In Encylopedia qf Plant Physioloqy. Physiology of’ Mouemwt. Mouement due to M~ckunicaland Ekctrical Stimuli and to Radiatioit (Edited by W. Ruhland). Springer, Berlin. Haupt, W. (1966) Intern. Reu. Cyrol. 19, 267-299. Lindes, D., B. Diehn and G. Tollin (1965) Rev. Sci. Instrum. 36, 1721-1725. Mast, S. 0. (191I ) Light and the Behavior of Organisms. Wiley. New York. Nultsch, W. (1970) In Photobiology oj’ Microorganisms (Edited by P. Halldal), pp. 213-252. Wiley, London. PTefier, W. (1904) Pflanzenphysiol. (transl. by A. J. Ewart) Clarendon, Oxford.

Phototactic response of Chlamydomonas to flashes of light. I. Response of cell populations.

Photochemistry and Phutuhrolugy, 1975. Vol. 21, pp. 351 354. Pergamon Press. Printed i n Great Britain PHOTOTACTIC RESPONSE OF e H u M Y m M o N A s...
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