PHOTOTACTIC RESPONSE OF CHLAMYDOMONAS TO FLASHES OF LIGHT –II. RESPONSE OF INDIVIDUAL CELLS

Abstract— –Video-microscope studies provide further evidence that Chlamydomonas can become oriented in response to a single short flash of light. Following a flash, 50% of the cells in a negatively phototactic population undergo a transient deflection in swimming path ('turn response'), 10% show a 'stop response', and 40% continue to swim straight ahead. The direction of turning is related to the direction of the stimulus; a majority of cells turn away from the flash source. Repetitive flashing at 60 per s elicits oriented swimming, indistinguishable from that observed with continuous light. Responses at the onset of repetitive flashing resemble single-flash responses, reinforcing the idea that response to a single flash corresponds to the initial stages of orientation to continuous light. A stop response sometimes occurs at the onset of orientation to repetitive flashing, but it is apparently not an essential component of orientation. The fact that only 60% of the cells turn or stop in response to a flash is consistent with the hypothesis that light direction is perceived by comparing light absorbed in one photoreceptive region at two instants in time (before and during the flash). The only cells to turn or to stop would be those in which the photoreceptor organelle is appropriately oriented at the instant of the flash.     

PHOTOTACTIC BEHAVIOR OF INDIVIDUAL CELLS OF CRYPTOMONAS SP. IN RESPONSE TO CONTINUOUS AND INTERMITTENT LIGHT STIMULI

Abstract— The phototactic response of cells of Cryptomonas sp. to stimulation with continuous or intermittent lateral light was determined by an individual cell method using photomicrography and videomicrography. The cells showed positive phototaxis under the conditions studied. The phototactic orientation of individual cells was induced most effectively by irradiation with light of 570 nm; blue light was less effective, and no orientation was found in red light. An intermittent stimulus regime with a long dark interval (250 ms) elicited a weaker phototactic orientation than did a regime with a short dark interval (63 ms) irrespective of the duration of light pulses (16, 250 and 1000 ms). The swimming rate was ca. 240 ums ^-1 and the rotation period ca. 450 ms in the dark, neither of which was greatly affected by stimulation with continuous or intermittent light. Neither step-up nor step-down photophobic responses were observed at the time of onset or removal of the light stimulus under the experimental conditions. The swimming direction of individual cells became gradually oriented toward the light source. Phototactic response was detectable within 4 s after the onset of light stimulation, reaching a saturation level after more than 30 s.     

CALCIUM CONTROL OF PHOTOTACTIC ORIENTATION IN Chlamydomonas reinhardtii: SIGN AND STRENGTH OF RESPONSE

Abstract— Chlamydomonas reinhardtii responds to a blue light stimulus by an oriented swimming (phototaxis) toward or away from the stimulus source. In this study it is established that the sign and strength of the phototactic response are a complex function of extracellular [Ca²⁺], stimulus fluence rate, time of analysis after onset of stimulation and light pretreatment. At very low extracellular [Ca²⁺] the response is weak and usually negative. At [Ca²⁺] close to the preconditioning level, phototactic response becomes stronger and positive. As [Ca²⁺] is raised further, the initial (2 s) response remains positive but the long term (20 s) becomes negative and very strong. At extremely high [Ca²⁺] the cells become immobile. This bimodal behavior suggests that two different mechanisms determine the direction of the turn. Data cannot be explained in terms of a simple model. The model which accounts for most of the details of the behavior is that of Kamiya and Witman (1984), which proposes that positive response is triggered by a transient increase in intracellular [Ca²⁺] and negative response by a decrease below unstimulated level of Ca²⁺, at least in the range of 10^-9-10^-6 M [Ca²⁺]. The strong negative orientation which follows an initial positive response above this level of [Ca²⁺], in these experiments, is best explained by an adaptation of the cells due to an increased (on average) intracellular [Ca²⁺].     

PHOTOMOVEMENT IN AN "EYELESS" MUTANT OF Chlamydomonas

The function of the stigma ("eyespot") in the green flagellate Chlamydomonas reinhardtii was investigated by comparing the photomovement of the wild-type alga with that of an "eyeless" mutant ( ey 627). Movements of individual cells in response to a blue-green stimulus light were recorded using a videomicroscope system and were analyzed using vectorial methods. Cells of the "eyeless" mutant were phototactic; at a high stimulus fluence rate, their swimming paths were directed away from the light source. Although the orientation of the mutant was not as strongly directional as that of the wild type, it was statistically significant. However, the swimming paths of the mutant cells were very erratic in the presence of the stimulus beam, undergoing frequent changes of direction. Despite the differences in their phototactic orientation, cells of mutant and wild type all showed a distinct step-up photophobic response at the onset of stimulation. These results are consistent with the hypothesis that the stigma plays an accessory role in phototaxis, either by shading the photoreceptor or by acting as a quarter-wave reflector.     

Peculiar properties of chlorophyll thermoluminescence emission of autotrophically or mixotrophically grown Chlamydomonas reinhardtii

The microalgae Chlamydomonas reinhardtii and Chlorella sp. CCAP 211/84 were grown autotrophically and mixotrophically and their thermoluminescence emissions were recorded above 0 °C after excitation by 1, 2 or 3 xenon flashes or by continuous far-red light. An oscillation of the B band intensity according to the number of flashes was always observed, with a maximum after 2 flashes, accompanied by a downshift of the B band temperature maximum in mixotrophic compared to autotrophic grown cells, indicative of a dark stable pH gradient. Moreover, new flash-induced bands emerged in mixotrophic Chlamydomonas grown cells, at temperatures higher than that of the B band. In contrast to the afterglow band observed in higher plants, in Chlamydomonas these bands were not inducible by far-red light, were fully suppressed by 2 μM antimycin A, and peaked at different temperatures depending on the flash number and growth stage, with higher temperature maxima in cells at a stationary compared to an exponential growth stage. These differences are discussed according to the particular properties of cyclic electron transfer pathways in C. reinhardtii.     

REVERSIBLE BLEACHING OF Chlamydomonas reinhardtii RHODOPSIN in vivo

Abstract— The effect of hydroxylamine on the phototactic activity of Chlamydomonas reinhardtii was investigated. The following results were obtained: (1) wild type cells, irradiated for 10 min with green light immediately after addition of 1 mM hydroxylamine, showed a 20 min transient loss of phototactic activity, (2) irradiation of cells, preincubated in the dark with 4 mM. hydroxylamine for 30 min, diminished the phototactic sensitivity permanently by more than 100-fold without loss of cell motility. (3) The phototactic sensitivity completely recovered within 3(1 min of the removal of hydroxylamin from carotenoid-containing cells or from carotenoid-negative cells upon addition of 11- cis or all- trans retinal. Our explanation is bleaching of rhodopsin by more than 99% and reconstitution by de novo synthesized or by added retinal.     

AN OSCILLATING SYSTEM REGULATING DEVELOPMENT OF PLANTS

Phytochrome conversion shifts the developmental pattern of dark-grown bean (Phaseolus vulgaris) seedlings. Red light was found to initiate rapid oscillations in a system that links illumination with subsequent growth. A single 8 s flash of red light increased the average leaf weight measured 24 h later by 8%. When total illumination was kept constant but the interval between two 4 s flashes was varied, the resulting leaf weight increase was not uniform but depended on the length of the interval between flashes and showed a series of deep minima followed by sharp maxima. Weight increase at the maximum was 60% greater than at the minimum. The minimum-maximum transitions recurred every 45 s for at least 22 min. Four s of far red light interposed between the two red flashes abolished the oscillation. Temperature between 15 and 30°C had no significant influence on the period of the oscillation but the period varied directly with the duration of the initial red flash. A 2 s initial flash resulted in a 35 s period, while 40 s of red light caused a 2 min period. Oat (Avena sativa) and radish (Raphanus sativus) seedlings were found to possess similar oscillating mechanisms of growth regulation. The lack of pronounced temperature effects as well as our other findings, suggest that this oscillating system may participate in the time measuring as well as growth regulating mechanisms by which phytochrome controls circadian periodicity and development.     

OSCILLATIONS OF THE TRIGGERED LUMINESCENCES OF ISOLATED CHLOROPLASTS PREILLUMINATED BY SHORT FLASHES*

Abstract— Four types of triggered luminescence of isolated lettuce chloroplast (HCl-induced, methanol-induced, sodium benzoate-induced and T-jump-induced) were examined after preillumination by a series (from 1 to 10) of short flashes. Oscillations were observed in the luminescence peaks, with a period of four flashes. These oscillations had maxima after the second and the sixth flash, similar to those of delayed light emission. The maxima were shifted forward two flashes by 50 μ M hydroxylamine, as in oxygen evolution, and were abolished by 5 μ M DCMU, as for delayed light. These results may show that the mechanism of triggered luminescence is influenced directly by the oxidation states ( S ₁) on the donor side of photo-system II.     

ACTIVATION KINETICS OF PHOTOSYNTHETIC OXYGEN EVOLUTION UNDER 20–40 NANOSECOND LASER FLASHES

Abstract— Chlorella samples, incubated for varying periods in darkness, were exposed to a series of 20–40 nsec flashes, spaced 15 sec apart, from a Q-switched ruby laser. A stationary Teflon-covered platinum electrode measured the microjet of oxygen produced by each flash. After a dark preincubation exceeding 3 min, at 23°C, little or no oxygen is evolved until the third flash in a sequence. The yields from subsequent flashes increase monotonically until a constant value is reached. If low levels of background light are supplied, or if the interval between series of flashes is decreased, oxygen is evolved on the second or even the first flash. Very similar results were obtained from analogous experiments with tailless 28 μsec flashes from a xenon flashtube. In particular, very little oxygen was evolved from the second flash following a long dark period whatever the spacing between flashes. This means that there are no systematic differences between the effects of saturating 20 nsec and 28 μsec light flashes on the activation processes during the first few flashes following a long dark period. No oscillations of flash yield with successive flashes were observed because of the long interval between flashes. These results are consistent with the idea that the reaction center of Photosystem II must undergo a dark process lasting considerably longer than 28 μsec before it can absorb a second photon.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

COMPARAISON ENTRE L''EMISSION D''OXYGENE ET L''EMISSION DE LUMINESCENCE A LA SUITE D''UNE SERIE D''ECLAIRS SATURANTS

Abstract— Luminescence of Chlorella cells has been measured after illumination by a series of short saturating flashes.
The intensity of luminescence is strongly dependent upon the number of flashes; luminescence is minimum after a single flash and maximum after a double flash illumination.
If l /_n is the intensity of luminescence measured 0.24 sec after the nth flash, the series l _n shows oscillations as a function of n. The series l _n is very similar to the series y _n, in which y is the amount of oxygen evolved by the nth flash, the term l _n corresponding to the term y _n+1. To interpret the oscillations of the series y _n, different models of system II photochemical centers have been proposed, one by ourselves and the other one by KOK et al. In these models the electron donor Z of photosystem 11 is supposed to have 2 or 4 levels of oxidation, the more oxidized state being the precursor of oxygen. These different oxidation states of the donor Z allow storage of the photic energy in the system. The correlation between the series y _n and l _n shows that at least one of the oxidized forms of the donor Z is a substrate for the luminescence reaction.     

Effect of preillumination on photomotile responses of the marine ciliate Fabrea salina

Fabrea salina is a marine ciliate that shows photomotile responses such as positive phototaxis and a step-down photophobic reaction. We found that preilluminated F. salina cells show a phototactic response significantly greater than that of dark-adapted cells when exposed to the same phototactic light stimulus. In particular, positive phototaxis is strongly enhanced by preillumination. This enhancement effect depends on the preillumination light irradiance, on the total preillumination dose, and on the duration of the dark interval between preillumination and the phototaxis measurement. Our results show that the determining factor is the total preillumination dose given to the sample. The enhancement effect shows an asymptotic behavior over a certain range of energy values (10-200 W/m2). Further, the effect is transient; after 120 s in the dark, the cells lose any memory of the preillumination, independent of the preillumination energy received. These results are tentatively discussed in terms of light-driven membrane potential or membrane channel conductances.     

PHOTOPHOBIC BEHAVIORAL RESPONSES OF EUGLENA IN A LIGHT INTENSITY GRADIENT AND THE KINETICS OF PHOTORECEPTOR PIGMENT INTERCONVERSIONS*

Abstract— Accumulation of Euglena gracilis in small illuminated regions called light traps is due to a phobic response to the diminished light intensity at the boundary of the region. The rate of such accumulation of cells was measured as functions of both the light intensity within the trap and the change of intensity at the boundary of the trap. The initial rate of accumulation of a population of cells was taken to be a direct measure of the phobic response of a single typical cell. The data indicate that the strength of the behavioral response in a single cell may be described as being proportional, to the rate of change of the amount of photochemically active form of a photoreceptor pigment molecule which can exist in two predominant forms.     

PHOTOBEHAVIOR OF EUGLENA GRACILIS: ACTION SPECTRUM FOR THE STEP-DOWN PHOTOPHOBIC RESPONSE OF INDIVIDUAL CELLS

Abstract—Light-induced behavioral responses of Euglena gracilis have been investigated in single cells by means of a video system coupled to an optical microscope. Light intensity-effect curves at different wavelengths in the near UV and visible range have been determined. From these curves the action spectrum for the step-down photophobic response of Euglena has been calculated. From a comparison with the results obtained using a population method by means of a phototaxigraph, it is concluded that a single photomotile reaction is responsible for cell accumulation, brought about by trapping in the light spot and possibly by phototaxis towards scattered light from organisms already in the light field.     

MODEL CONSIDERATIONS OF THE LIGHT CONDITIONS IN NONCYLINDRICAL PLANT CELLS

Abstract— A geometric model of a noncylindrical plant cell has been constructed using optical and geometrical parameters obtained for the cells of Funaria hygrometrica and Lemna trisulca leaves. The distribution of the incident light intensity and the absorption profile in the monomolecular photoreceptor layer on the walls of the model cell irradiated with the parallel beam have been calculated. Calculations were performed for two surrounding media with refractive indices of 1.33 (water) and 1.00 (air). Various chloroplast arrangements were considered. The course and kinetics of the phototactic movements of Funaria and Lemna chloroplasts have been discussed on the basis of the results obtained. The best agreement between the theoretical and experimental data is obtained by assuming that the transition moments at 450 nm of the dichroic photoreceptor molecules are oriented parallel to the surface of the cell wall.     

Optical and electric signals from dried oriented purple membrane of bacteriorhodopsins

All the intermediates of the bacteriorhodopsin photocycle are excitable with light of suitable wavelength. This property might regulate the activity in the cells when they are exposed in the nature to high light intensity. On the other hand this property is involved in many applications. In this study the ground state and M intermediate of dried oriented samples of wild-type bacteriorhodopsin and its mutant D96N were excited with 406 nm laser flashes. Substantial M populations were generated with quasi-continuous illumination. The decay of the absorption of M intermediate had three components: their lifetimes were very different for laser flash and quasi-continuous illuminations in cases of both bacteriorhodopsin species. The optical answer for the excitation of M intermediate had a lifetime of 2.2 ms. Electric signals for M excitation had large fast negative components and small positive components in the 100 μs time domain. The results are expected to have important implications for bioelectronic applications of bacteriorhodopsin.     

KINETIC ANALYSIS OF THE INFLUENCE OF HYDROSTATIC PRESSURE ON BIOLUMINESCENCE OF GONYAULAX POLYEDRA

Abstract— Hydrostatic pressure was used as a probe to examine control mechanisms of bioluminescence in Gonyaulax polyedra. The initial effect of a pressure increase step is both to increase the intensity of the continuous light emission (glow) of the entire cell population and to increase the frequency of discrete flashes arising from single cells. Following the pressure application, however, the glow does not merely attain a new level but rather goes through various transient changes, whereby the kinetics of these changes are faster with a given higher pressure. A qualitative fit to several aspects of the pressure induced glow kinetics was generated by a simple reaction rate theory model. The effect of pressure upon the circadian rhythm control of bioluminescence was also investigated with the result being that no significant influence was observed under the experimental conditions.     

THE ACTION SPECTRUM OF DAPHNIA MAGNA (CRUSTACEA) PHOTOTAXIS IN A SIMULATED NATURAL ENVIRONMENT

Abstract— The action spectrum of phototaxis in Daphnia magna (Crustacea) was measured in a chamber which simulated a natural angular distribution of underwater light. A 17% step-down in irradiance was used to stimulate the phototactic response at all wavelengths and irradiances tested. Peaks in the spectral response curves depended on the fluence rate to which the zooplankton were acclimated. The wavelength of maximum response (Z_max) shifted from yellow-green at the highest acclimation fluence rate (5.1 × 10⁻² Wm⁻²) to blue-violet at moderate rates. At low acclimation fluence rates, the blue-violet maximum was retained and another maximum developed in the red. At the lowest fluence rate (1.6 × 10⁻⁵ Wm⁻²), the blue-violet and red maxima were lost and another maximum developed in the near ultraviolet. The action spectrum indicates the presence of three, and possibly four, photopigments with Z_max, at ∼405, 440, 570 and 690nm. The 440 and 690nm maxima may belong to the same photopigment; however, this was not tested. Changes in zooplankton swimming speed, caused either by large changes in irradiance or by mechanical stimuli, were accompanied by changes in the strength of the phototactic response to the −17% stimulus at any irradiance level for white and monochromatic light, and indicated the presence of a mechanism connecting swimming speed and photosensitivity.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

INDUCTION OF SEED GERMINATION IN Lactuca sativa L. BY NANOSECOND DYE LASER FLASHES*,†

Abstract— A 15 ns, tunable dye laser was used to induce germination of the photoblastic seeds of Lactuca sativa. One red laser flash in the range from 620 to 690 nm was sufficient to increase germination significantly above the dark level. Repeated flashes, however, were necessary to saturate the physiological response. The wavelength dependence for induction of germination differed for single and repetitive flashes. After saturating far-red irradiation, the effect of single-flash induction was a function primarily of the absorption spectrum of P_r. In addition, the establishment within the lifetime of a flash of a photochromic system between the red absorbing form of phytochrome (P_r) and the sum of photoreversible intermediate forms (ΣI₇₀₀) contributes to this wavelength dependence at high fluence rates. This photochromic system is assumed to be shifted significantly toward P_r by wavelengths 660 nm. Similarly, a strong double-flash effect, which is seen as an increase in effectiveness when a given total fluence is provided by two consecutive flashes rather than by one flash only, is restricted to those wavelengths that considerably shift the photochromic system P_r?ΣI₇₀₀ toward P_r. Finally, the saturation level produced by a series of laser flashes depends, additionally, on absorption by P_fr.