Effect of pronase treatment on step-down and step-up photophobic responses in Euglena gracilis

Pronase-treated cells of Euglena gracilis Z show no discernible ultrastructural effects on the photoreceptor apparatus; however, there are physiological effects on swimming speed and on step-up and step-down photophobic responses, especially the latter. Pronase acts differently on the two photophobic responses: the step-down response is completely inhibited after 2 hr., whereas inhibition of the step-up response occurs in only 50% of the cells even after 24 hr. The effects are fully reversible, with step-up recovery quite rapid and step-down recovery considerably slower.     

Discovery of signaling effect of UV-B/C light in the extended UV-A/blue-type action spectra for step-down and step-up photophobic responses in the unicellular flagellate algaEuglena gracilis

Summary Cultures of unicellular algal flagellateEuglena gracilis grown in different conditions were subjected to action spectroscopy for step-down and step-up photophobic responses, respectively. The spectral region was extended into the UV-B/C as well as in the UV-A and visible regions with the Okazaki Large Spectrograph as the monochromatic light source. The photophobic responses of the cells were measured with an individual-cell assay method with the aid of a computerized video motion analyzer. In the UV-A and visible regions, the shapes of the action spectra were the so-called UV-A/blue type. In the newly studied UV-B/C region, new action peaks were found at 270 nm for the step-down response and at 280 nm for the step-up one. The absorption spectrum of flavin adenine dinucleotide (FAD) appeared to fit the action spectrum for the step-up response, whereas the shape of the step-down action spectrum, which has a UV-A peak (at 370 nm) higher than the blue peak (at 450 nm), appeared to be mimicked by the absorption spectrum of a mixed solution of 6-biopterin and FAD. These observations might also account for the fact that the UV-B/C peak wavelength at 270 nm of the action spectrum for the step-down response is shorter by 10 nm than the action spectrum for the step-up response at 280 nm.Abbreviations FAD flavin adenine dinucleotide - FWHM spectral full width at half maximum - NIBB National Institute for Basic Biology - OLS Okazaki Large Spectrograph - PFB paraflagellar body - UV-A ultraviolet light of spectral region between 320 and 400 nm - UV-B/C ultraviolet light of spectral region between 190 and 320 nm     

Effects of sequential stimuli on Halobacterium salinarium photobehavior.

We analyzed the motor photoresponses of Halobacterium salinarium to different test stimuli applied after a first photophobic response produced by a step-down of red-orange light (prestimulus). We observed that pulses given with a suitable delay after the prestimulus produced unusual responses. Pulses of blue, green, or red-orange light, each eliciting no response when applied alone, produced a secondary photophobic response when applied several seconds after the prestimulus; the same occurred with a negative blue pulse (rapid shut-off and turning on of a blue light). Conversely, no secondary photophobic response was observed when the test stimulus was a step (a step-up for red-orange light, a step-down for blue light) of the same wavelength and intensity. When the delay was varied, different results were obtained with different wavelengths; red-orange pulses were typically effective in producing a secondary photophobic response, even with a delay of 2 s, whereas the response to a blue pulse was suppressed when the test stimulus was applied within 5 s after the prestimulus. The secondary photophobic response to pulses was abolished by reducing the intensity of the prestimulus without affecting the primary photophobic response. These results, some of which were previously reported in the literature as inverse effects, must be produced by a facilitating mechanism depending on the prestimulus itself, the occurrence of reversals being per se ineffective. The fact that red-orange test stimuli are facilitated even at the shortest delay, whereas those of different wavelengths become effective only after several seconds, suggests that the putative mechanism of the facilitating effect is specific for different signaling pathways.     

Step-up photophobic response in the ciliate,Stentor coeruleus

The avoidance by Stentor coeruleus of a light trap is caused by a step-up photophobic response. The phobic response invariably consists of a delay of about 200 ms, a stop response, a turn to one side, and resumption of swimming in the new direction. After this the cells enter a refractory period of 1–3 s following a phobic response, during which they will not give a second response. Phobic responses can be elicited by spatial and temporal increases in light intensity. The action spectrum for the step-up photophobic response resembles the absorption spectrum of stentorin, the proposed photoreceptor pigment, and of its chromophore, hypericin.The phobic response is specifically inhibited by the protonophorous uncouplers TPMP⁺ and FCCP but not by the ionophores gramicidin and A23187. Since the uncouplers block light-induced membrane potential changes at the same concentrations, it has been proposed that the primary photoreception causes a light-induced potential change, which in turn, induces a motor response.Abbreviations TPMP⁺ triphenyl methyl phosphonium bromide - FCCP carbonylcyanide p-trifluoromethoxy-phenylhydrazone     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Action Spectra for Step-Up Photophobic Response in Blepharisma

ABSTRACT The cells of Blepharisma which possess red pigment (blepharismin) show step-up photophobic response (temporal ciliary reversal induced by a sudden increase in light intensity). Bleaching of the cells by cold shock raised a threshold light intensity for the response, Oxidation of red pigment that produced blue pigment did not raise the threshold for the response. The action spectrum for the step-up photophobic response of the cells which possess normal red pigment had peaks at about 580, 540 and 490 nm, a value which coincided with peaks of an absorption spectrum of the red pigment. The absorption spectrum of oxidized pigment (blue pigment) shifted 20 nm toward infrared light. The action spectrum for the response of the cells which possess blue pigment also shifted 20 nm toward infrared light. Results suggest that red pigment might be involved in the step-up photophobic response. Key words. Blepharismin, ciliary reversal, photoreceptors, photoresponse.     

Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.     

Dependence of the photophobic response of the blue-green alga,Phormidium uncinatum,on cations

It is suggested that photophobic responses caused by a sudden step-down in light intensity require the presence of cations in the blue-green alga, Phormidium uncinatum.Drastic removal of cations abolishes the phobic response, which recovers after addition of Ca²⁺ ions. Calcium can be substituted for partially by other cations with an effectivity following the sequence Ca>Mg>Na>Ba>Co=0. During the photophobic response there is a 25% increase in ⁴⁵Ca binding by the cells related to a step-down in light intensity. Three seconds after a light-dark transition there is a sharp increase in the binding of labelled calcium, followed by a subsequent release.Flushing the filaments with high cation concentrations, esp. calcium causes a reversal of movement in the absence of a light stimulus similar to a photophobic reversal. This stimulus could trigger the same sequence of events in the transduction chain bypassing the primary photoresponse.Abbreviations EDTA Ethylene diaminetetraacetic acid - EGTA ethylene glycol-bis (2-aminoethylether) N,N tetraacetic acid     

Photoresponse and Learning Behavior of Ascidian Larvae, a Primitive Chordate, to Repeated Stimuli of Step-Up and Step-Down of Light

Ascidians are lower chordates and their simple tadpole-like larvae share a basic body plan with vertebrates. Newly hatched larvae show no response to a stimulus of light. 4 h after hatching, the larvae were induced to swim upon a step-down of light and stop swimming upon a step-up of light. At weaker intensity of light, the larvae show the same response to a stimulus after presentation of repeated stimuli. When intensity of actinic light was increased, the larvae show sensitization and habituation of the swimming response to a stimulus after repeated stimuli of step-down and step-up of the light. Between 2 h 20 min and 3 h 40 min after hatching the larvae did not show any response to the first stimulus, but after several repeatedstimuli they show swimming response to a step-down of light. A repeated series of stimulus cause sensitization. Between 4 h and 7 h after hatching, the larvae show photoresponse to the first stimulus, but after several repetition of the stimuli, the larvae could not stop swimming to a stimulus of a step-up of the actinic light. A repeated series of stimulus cause greaterhabituation. Both sensitization and habituation depend upon intensity ofactinic light.     

Control by Ammonium Ion of the Change from Step-Up to Step-Down Photophobically Responding Cells in the Flagellate Alga Euglena gracilis

The regulation between step-down and step-up photophobic responses,resulting in photoaccumulation of the cells in an actinic lighttrap or cells' avoidance from an excessive illumination, iscrucially important for the survival of phototrophic organismssuch as Euglena gracilis. As for the factors involved in thisregulation in Euglena gracilis, we for the first time reporthere that ammonium ion specifically enhances step-down photophobicresponse, together with the effects of L-methionine-DL-sulfoximine(L-MSO), an inhibitor of ammonium assimilation, to specificallyenhance step-up photophobic response. The apparent positivecorrelation between the degree of greening and the step-downphotophobic response did not seem to reflect real causal relationshipin view of the results with effects of gabaculine, an inhibitorof -aminolaevulinic acid (-ALA) formation. The transmissionof stigma and step-down appearance did not show any correlationeither, in contrast to a previous assumption by other authors.Cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis,suppressed step-down appearance and enhanced step-up appearance,probably suggesting an involvement of some (newly synthesized)protein(s) specifically in the step-down photosignal detectionand/or signal transduction process(es). (Received August 18, 1998; Accepted December 3, 1998)     

Photomovement in Dunaliella salina: Fluence rate-response curves and action spectra

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450–460 nm. These action spectra are not compatible with the hypothesis that flavoproteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak a 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.Diagrams of the optical set-ups used for measuring the responses at the single-cell level and of the plans for building the phototaxometer described in this paper are available to the interested readerWe thank Mr. M. Kubota for a tremendous amount of technical assistance and Mr. R. Nagy for building the phototaxometer. We thank T. Kondo, Professor H. Imaseki and the members of the Laboratory of Biological Regulation, NIBB, for their help and support in various aspects of this research. This research was supported, in part, from grants from the Okazaki Large Spectrograph (Project Nos. 86-535, 87-518, 88-523), the Japanese Society for the Promotion of Science, and the College of Agriculture and Life Sciences at Cornell University to R. W.     

Light-adaptation in the photophobic response by Stentor coeruleus

Effects of preillumination on photophobic response (light-adaptation) and recovery of the photophobic sensitivity in the dark (dark-adaptation) in Stentor coeruleus were examined. When the cells were preilluminated with white light of 7.80 W/m² for 2 min, the fluence-rate response curve of photophobic response was shifted toward higher light intensities by half an order of magnitude compared to the one without preillumination. Preillumination with a higher light intensity resulted in a further shift of the fluencerate response curve. An action spectrum for light-adaptation showed a primary peak at 610 nm and secondary peaks at 540 and 480 nm which are almost identical to the peaks observed in the photophobic action spectrum.The light-adapted cells showed a recovery of their photophobic sensing ability following dark treatment. Dark-adaptation resulted in total recovery of photophobic sensing ability in 8 minutes for the most cases examined.     

Anion sensitivity of motility and step-down photophobic responses of Euglena gracilis

The motility and step-down photophobic responses of Euglena are influenced by inorganic and organic anions. Persistent motility (with Ca²⁺, Mg²⁺ and K⁺ present) is supported with chloride or sulfate but not with acetate, nitrate or propionate as the only added anions. Cells in media containing acetate displayed a cell aggregation (clumping) behavior that was both red light sensitive and, under some conditions, was accompanied by suppression of the step-down photophobic response. Addition of sodium salts (Cl^-, SO ₄ ^2- , acetate or propionate) to cells in Cl^- or SO ₄ ^2- based media had differential effects on the duration of the step-down photophobic responses induced by blue light removal: anions alter the response. In addition, cells in all Cl^- containing media showed constant photophobic response duration following repeated stimulation. Cells in some SO₄ ^2- containing media, however, showed response summation to repeated stimulation. This latter effect was reversible and was overcome by the addition of chloride anions.     

The ratio of extracellular Ca2+ to K+ ions affects the photoresponses in Stentor coeruleus

1. Stentor coeruleus exhibits negative phototaxis (due to phototactic orientation response) and step-up photophobic response (avoiding reaction) to visible light. 2. The effect of Ja-value ([K+]/[Ca2+]1/2) and calcium ion concentration of the surrounding medium on the photoresponses in Stentor were studied. 3. The both types of photoresponses in Stentor are greatly affected by the Ja-value. A higher Ja-value medium suppressed the step-up photophobic response of Stentor, whereas the organism showed a higher degree of phototactic orientation response in higher Ja-value solutions. 4. The effect of the Ja-value on the step-up photophobic response was opposite to that on the phototactic orientation response. 5. With increasing calcium concentration but at a constant Ja-value, the number of Stentor showing the step-up photophobic response increased, whereas the phototactic orientation response of Stentor was suppressed at higher Ca2+ concentrations. 6. The effect of the calcium concentration on the photophobic response was also opposite to that on the phototactic orientation response, as in the case of Ja-value effect.     

Deuterium oxide (D2O) enhances the photosensitivity of Stentor coeruleus.

Stentor coeruleus exhibits negative phototaxis and step-up photophobic response (avoiding reaction) to visible light (maximum at 610-620 nm in both responses). In the presence of deuterium oxide (D2O) the step-up photophobic response was markedly enhanced, whereas the phototactic orientation response was inhibited. The induction time for the step-up photophobic response was longer in D2O than in H2O, and the duration of ciliary reversal for the response was also longer in D2O than in H2O, indicating that certain steps of the sensory transduction chain are subject to solvent deuterium isotope effects. The enhancement of the step-up photophobic response in D2O was canceled by LaCl3, while the inhibition of the phototactic orientation response in D2O was partially removed by LaCl3, even though LaCl3 did not affect the phototactic orientation response. These results suggest that the sensory transduction mechanisms for the two photoresponses are different, although the photoreceptors (stentorin) are the same.     

Photobehaviour of Paramecium bursaria infected with different symbiotic and aposymbiotic species of Chlorella

The endosymbiotic unit of Paramecium bursaria and Chlorella spec. shows two types of photobehaviour: 1) A step-up photophobic response which possibly depends on photosensitive agents in the ciliate cell itself — as is also shown by alga-free Paramecium bursaria - and can be drastically enhanced by photosynthetic activity of symbiotic algae; and 2) a step-down photophobic response. The step-down response leads to photoaccumulation of green paramecia. Both types of photobehaviour in Paramecium bursaria do not depend on any special kind of algal partners: The infection of alga-free Paramecium bursaria with different Chlorella species results in new ciliatealgae-associations. They are formed not only by combination of the original symbiotic algae with their host, but also by infection with other symbiotic or free-living (aposymbiotic) chlorellae, respecitively. Systems with other than the original algae are not permanently stable — algae are lost under stress conditions — but show the same types of photobehaviour. Photoaccumulation in general requires algal photosynthesis and occurs only with ciliates containing more than fifty algae/cell. It is not mediated by a chemotactic response to oxygen in the medium, since it occurs at light fluence rates not sufficient for a release of oxygen by the symbiotic system, e.g., below its photosynthetic compensation point. Photoresponses can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Sensory transduction does not depend on any special symbiotic features of the algae, e.g., sugar excretion. The participation of oxygen in the Paramecium cell, of its cytoplasmic pH and of ions released or taken up by endosymbiotic algae in sensory transduction is discussed.     

Photomovement in Cyanophora paradoxa

Photomovement has been studied in the symbiontic association of the colorless flagellate, Cyanophora paradoxa Korschikoff with the cyanelles, Cyanocyta korschikoffiana. There is no phototactic orientation in this organism, but a photokinetic effect. In addition, the cells show a pronounced step-up photophobic response (however no or only a weak step-down response). The phobic response is mediated by a subset of the photosynthetic pigments located in the symbiontic cyanelles. It is linked to the noncyclic photosynthetic electron transport chain but it is independent of the photosynthetic generation of a proton gradient and the ATP synthesis linked to it.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzo quinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Phototaxis and photophobic responses in Stentor coeruleus action spectrum and role of Ca2+ fluxes

Negative phototactic orientation, step-up photophobic responses and light-induced action potentials have been studied in the ciliate Stentor coeruleus. A resolved action spectrum, based on fluence rate-response curves, is consistent with stentorin as the photoreceptor. Calcium flux blockers prolong the response time for ciliary stop and reversal and inhibit step-up photophobic responses. Drugs believed to affect the membrane-bound calcium pump likewise inhibit phobic responses. On the other hand, α-phosphatidic acid promotes Ca²⁺-influx and enhances the photophobic sensitivity of the organism, thus providing an unambiguous evidence for the role of Ca²⁺ influx. A change in the response time decreases the degree of phototactic orientation, indicating that negative phototaxis in this organism is brought about by subsequent phobic responses of individual rows of cilia as the associated photoreceptor granules experience an increase in light intensity when the organism rotates during forward locomotion in lateral light.     

Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: simultaneous measurements of phototactic and photophobic responses

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-trans retinal was more effective than 11-cis retinal to regenerate both photobehavioral responses. Analogs having locked 11-cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all-trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13-14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.     

Primary stages in photosignal transduction leading to step-up photophobic response in the unicellular eukaryote Blepharisma japonicum

In order to elucidate the primary stage in the blepharismin phototransduction pathway, changes in the molecular structure of light-exposed blepharismins and oxyblepharismins, were examined. When exposed to light, blepharismins (pink form) were converted into oxyblepharismins (blue form) or dissociated into stentorins/p-hydroxybenzaldehyde with an O2-requiring process, whereas light-exposed oxyblepharismins were not dissociated into stentorins/p-hydroxybenzaldehyde. Since both blepharismins and oxyblepharismins can activate the phototransduction chain leading to the step-up photophobic response presumably through the same pathway, dissociation of p-hydroxybenzaldehyde may not be involved in signal transduction. The fact that the step-up photophobic response requires O2, and both blepharismins and oxyblepharismins produce hydroxyl (OH) radicals probably derived from O2 implies that OH radicals may activate the photosignalling pathway. The step-up photophobic response was not suppressed by a spin trapping reagent for hydroxyl radicals. Other possible primary responses leading to the step-up photophobic response are discussed.