Green- and blue-light-mediated chloroplast migration in the centric diatomPleurosira laevis

Protoplasma (1998) 203:214-220

PROTOPLASMA �9 Springer-Verlag 1998 Printed in Austria

Green- and blue-light-mediated chloroplast migration in the centric diatom Pleurosira laevis

T. Furukawa ~, M. Watanabe 2, and I. Shihira-Ishikawa 1,*

1Department of Biology, Tokyo Gakugei University, Koganei-shi, Tokyo and 2National Institute for Basic Biology, Okazaki, Aichi

Received December 15, 1997 Accepted April 2, 1998

Summary. The existence of two photoreceptors regulating chloroplast orientation was found in the centric diatom Pleurosira laevis.

Chloroplasts migrate through the transvacuolar cytoplasmic strands according to the light conditions. Weak white light of less than 46 ~tmol/m 2 �9 s (10 W/m 2) induces chloroplast movement to the cortical cytoplasm, which is located next to the plasma membrane (dispersion), while intense white light of more than 92 ~tmol]m 2. s (20 W/m 2) induces chloroplast movement towards the nucleus, which is situated in the center of the cell (assemblage). Chloroplast dispersion was maintained as long as the ceils were irradiated with weak white light. Conversely, chloroplast assemblage under intense white light was transient and the chloroplasts were released from assemblage after 15 rain. Action spectra determined with the Okaza- ki Large Spectrograph revealed that the weak white light receptor and the intense white light receptor are characterized by 540 nm and 450 nm optima, respectively.

Keywords: Blue light; Chloroplast orientation; Diatom; Green light; Light fluence rate.

Introduction

Light-mediated chloroplast reorientation has been widely reported in higher plants and algae (Haupt 1965, 1982; Nagai 1993). In the diatoms Biddulphia pellucida (Senn 1919), Ditylum blightwellii (Chen and Li 1991), and Lauderia borealis (Keifer 1973), the chloroplast migration was found to be dependent mainly on the light-dark cycles. Pleurosira laevis (Ehr.) Compare (Compare 1982) is a fresh water diatom which forms filamentous colonies. Each cell, about 100 ~tm in length, is cylin- drical. The nucleus is situated in the center of a large

*Correspondence and reprints (present address): Marine Biotechnol- ogy Institute, Shimizu, Shizuoka 424-0037, Japan.

central vacuole. Disk-like chloroplasts, about 200 per cell, are distributed in the cortical cytoplasm, when the cells are illuminated by weak light, whereas under intense illumination the chloroplasts migrate towards the nucleus through transvacuolar strands (Shihira- Ishikawa and Ohsu 1995). Chloroplast migration towards the nucleus in P. laevis can also be induced by mechanical stimulation and in this case Ca 2+ influx through Ca 2+ channels may trigger chloroplast assemblage (Makita and Shihira-Ishikawa 1997). To characterize the photoreceptors which mediate chloroplast migration in P. laevis, we studied the effective light wavelengths and fluences. We found that two different photoreceptor systems are involved: one is green-light-regulated and controls chloroplast dispersion, while the other is UV-A/blue- light-regulated and controls chloroplast assemblage.

Material and methods Cells and culture conditions

Pleurosira laevis was collected from Tamagawa river in Tokyo. It grows in water at depths between 5 and 30 cm. A unialgal culture was maintained in BBM culture medium containing 2.94 mM NaNO3, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaC1, 0.30 mM MgSO4 �9 7H20, 0.18 mM Na2SiO3.9H20, 0.18 mM H3BO3, 0.17raM CaC12.2H20, 0.13 mM Na2EDTA, 55.25 ~tM KOH, 30.67 gM ZnSO4 �9 7H20, 29.65 ~M Co(NO3)2 �9 6H20, 18.00 ~M H2SO4, 17.59 ~tM FeSO4 �9 7H20, 7.28 ~tM MnC12 �9 4H20, 6.29 ~M CuSO4 �9 5H20, 4.93 ~M MOO3, 29.65 ~tM thiamine HC1, and 0.07 ~tM VBtz (Bold and Wynne 1978) under illumination with daylight fluorescent lamps, 500 lux, L : D = I2 h : 12 h, at 22 ~ The cells used for the experiments had been maintained in the labora- tory for less than 4 weeks after collection.

T. Furukawa et al.: Two photoreceptors in Pleurosira laevis 215

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White-light irradiation

Cells were placed on a hole-slideglass with culture medium and were irradiated with a 20 W daylight fluorescent lamp (FL20SD Nation- al). The fiuence rates were adjusted by changing the distance between the samples and the fluorescent lamp. Light fluence rate was measured with a multifunction photometer (Conmic-100B; Concorse Co. Ltd., Tokyo). Before experimental irradiation, the cells were kept in darkness for 3 h and, as a result, all of the chloroplasts in the cells were distributed randomly. Chloroplast migration in each cell was observed every 15 rain with a stereomicroscope (SZH-ILBB Olympus). More than 30 cells were used for every irradiation experiment. Effectiveness of light on chloroplast migration was expressed as the ratio of cells with chloroplast assemblage or dispersion to the total cells observed.

cells on average were observed for each irradiation experiment and the same experiments were repeated at least three times.

Absorption spectrum

For obtaining an absorption spectrum of the cells, the cell suspen- sion, about 4 x 104 cells/ml was placed in a quartz cuvette and measured directly in a spectrophotometer (228A Hitachi; Hitachi Ltd., Tokyo). To obtain an absorption spectrum of an extract of cells, 100% methanol was used for extraction. The cell extract was measured with the same method as the intact cells.

Results

Patterns of chloroplast migration

Chloroplasts were randomly distributed when the cells were kept in darkness for more than 1 h (Fig. 2 a, a'). When the cells were exposed to weak light of fluence rates less than 46 ~tmol/m 2 �9 s (10 W/m2), all of the chloroplasts migrated to the periphery of the cell (dispersion), passing through the transvacuolar strands (Fig. 2 b, b'). When the cells were exposed to intense light of fluence rates above 92 txmol/m 2. s (20 W/m2), all of the chloroplasts migrated towards the nucleus at the center of the cell (assemblage) (Fig. 2 c, c'). In weak light the chloroplasts began to move immediately after the start of irradiation and the per- centage of cells with chloroplast dispersion gradually increased, whereas in intense light the chloroplasts did not move during the first 10 rain of irradiation but then suddenly started to move towards the nucleus. The speed of chloroplast migration in transvacuolar cytoplasmic strands was ca. 3.6 ~tm/min in chloroplast dispersion and ca. 60 ~tm/min in assemblage.

Monochromatic-light irradiation

The Okazaki Large Spectrograph at the National Institute for Basic Biology (Watanabe et al. 1982, Watanabe 1991) in Okazaki, Japan was used for monochromatic irradiation at 10 nm interval. Samples were preincubated in darkness for 3 h and placed on the objective stage of the inverted microscope (Fig. 1). A partially reflecting mirror (30% neutral density filter) (BK-7, glass plate eva- porated with inconnel alloy; Fujitok, Tokyo) and appropriate neutral density filters were inserted into the light path to provide the monochromatic light and to adjust the fluence rate, respectively. The fluence rate of monochromatic light was measured just in front of the partially reflecting mirror by means of a photon density meter (HK- 1, custom-made by the Riken Institute for Physical and Chemical Research, Saitama, Japan) (Hashimoto et al. 1982), and the exact fluence rate at the sample stage was obtained by multiplying the above value by a calibration factor of 0.9 which had been empirically determined at monochromatic light, 600 nm and 650 nm. The percentages of cells with either pattern of chloroplast distribution were obtained 15 rain after the start of irradiation by video recording the images of cells which were observed with blue light at 50 W/m 2 for several sec- onds. This pulse of blue light did not have any effect on the chloroplast migration during the irradiation period for recording. Fifteen

Dependency of chloroplast migration on fluence rate of white light

The time course of the chloroplast migration was studied at various fluence rates (Fig. 3). After the cells were maintained in darkness for 3 h, they were irradiated with light at different fluence rates. At fluence rates of 0.46, 4.6, and 46 btmol/m 2 �9 s (0.1, 1, and 10 W/m2), only chloroplast dispersion was induced (Fig. 3 a-c). Conversely, chloroplast assemblage was induced at fluence rates of 92 gmol/m 2 �9 s (20 W/m2), although it was subsequently followed by dispersion after 15 rain (Fig. 3 d). Such transient chloroplast assemblage was also observed at 140, 180, and 230 btmol/m 2. s (30, 40, and 50 W/m 2) (Fig. 3 e-g). Under 230 ~tmol/m 2. s (50 W/m 2) irradiation, chloroplast assemblage was maintained for more than 15 rain, and dispersion was not induced even after assemblage decreased (Fig. 3 g).

216 T. Furukawa et al.: Two photoreceptors in Pleurosira laevis

Fig. 2. Patterns of chloroplast migration, a and a' Chloroplasts without migration. The chloroplasts are distributed randomly in the cell. b and b' Chloroplast dispersion. All of the chloroplasts are distributed in the cortical cytoplasm, e and c' Chloroplast assemblage. Most of the chloroplasts are located close to the nucleus, lying on top of each other

Wavelength dependency of chloroplast migration

To determine the wavelength dependency of chloroplast dispersion and assemblage, we utilized monochromatic lights with the Okazaki Large Spectro- graph at the National Institute for Basic Biology. We used fluence rates for chloroplast dispersion and assemblage of 9 btmol/m 2. s and 18 btmol/m 2 �9 s, respectively, based on the preliminary experiment using color filters. For chloroplast dispersion, a spectrum with a single peak at 540 nm was observed (Fig. 4 a). For chloroplast assemblage, light of wavelengths shorter than 480 nm was most effective, with a major peak at 450 nm (Fig. 4 b).

Dependencies of chloroplast migration on monochromatic-light fluence rate The monochromatic-light fluence rate dependency of chloroplast dispersion at 540 nm and chloroplast assemblage at 450 nm was investigated (Fig. 5). Cells preincubated in darkness for 3 h were exposed to either 540 nm or 450 nm light for 15 min with different fluence rates. At 540 nm, 9 btmol]m 2 �9 s was most effective for induction of chloroplast dispersion, and above this fluence rate the higher the fluence rates the smaller was the number of cells displaying chloroplast dispersion (Fig. 5 a). At 450nm, chloroplast assemblage increased with increasing fluence rate and the maximum ratio was obtained at 36 btmol/m 2 �9 s (Fig. 5 b).

T. Furukawa et al.: Two photoreceptors in Pleurosira laevis

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Test for reciprocity

Cells preincnbated in darkness for 3 h were exposed to either 540 nm or 450 nm light at different fluence rates and for different periods of time (Fig. 6). The periods of irradiation were approximately 2 min, 4 min, 8 min, and 15 min. Cells were kept in darkness

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after each period of irradiation and chloroplast migration was observed 15 rain after the onset of irradiation. In control experiments, it was confirmed that the dark period after irradiation did not influence chloroplast migration. Almost 90% of chloroplast dispersion and chloroplast assemblage were induced by irradiation at

218 T. Furukawa et al.: Two photoreceptors in Pleurosira laevis

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9 ~tmol/m 2 �9 s for 15 min, and at 36 gmol/m 2. s for 4 min, respectively (Fig. 6). The fluence required for this maximum frequency of chloroplast dispersion (8.1 mmol/m 2. s) was the same as for chloroplast assemblage. However, the maximum frequency of chloroplast dispersion was induced by weak and long- term irradiation of 540 nm whereas chloroplast assemblage was induced by intense and short-term irradiation of 450 nm light.

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P. laevis, a Whole cells; b methanol extract

Absorption spectra of whole cells and methanol extract

The absorption spectra of the whole cells (cell sus- pension) and the methanol extract of P. laevis are shown in Fig. 7. In whole cells (Fig. 7 a), a peak at 674 nm and shoulders at 342 nm, 378 nm, 416 nm, 437 nm, and 637 nm are attributed to chlorophyll a. The shoulders at 461 nm, 585 nm, and 618 nm are attributed to chlorophyll c. Four shoulders at 452 nm, 470 nm, 495 nm, and 535 nm are attributed to fucoxanthin. In methanol extracts (Fig. 7 b), two peaks at 3 3 8 n m and 5 5 7 n m and shoulders at 391nm, 420 nm, and 634 nm are attributed to chlorophyll a and the shoulders at 584 nm and 616 nm are attributed to chlorophyll c. The small shoulders of fucoxanthin in intact cells were lost in the methanol extract.

Discussion

Light-mediated chloroplast migration from the cortical cytoplasm to the nucleus-associated cytoplasm has been reported in three other centric diatoms, Bid- dulphia pellucida (Senn 1919), Ditylum bIightwellii (Anderson and Sweeney 1978, Chen and Li 1991), and Lauderia borealis (Keifer 1973). In these experiments, darkness was a signal for chloroplast assemblage around the nucleus. However, in Pleurosira laevis, when the cells were placed in darkness, the chloroplasts were distributed randomly inside the cell. This type of chloroplast arrangement in darkness is similar to Vallisneria gigantea (Izutani et al. 1990). Therefore in all of the experiments reported in the current article, cells were preincubated in darkness in order to randomly distribute the chloroplasts within the cytoplasm prior to irradiation. The action spectrum for chloroplast dispersion in P. laevis displayed a single peak at 540 nm. In the absorption spectra of whole cells and methanol extract, an absorption peak at 540 nm was not detected (Fig. 7). The slight increase of optical density at 535 nm in whole cells was not detected in methanol extract, suggesting that absorption at 535 nm was not attributable to the green-light receptor but to fucoxanthin together with other small peaks, 452 nm, 470 nm, and 495 nm (Kato et al. 1989). The light receptor for chloroplast dispersion is not likely to be a cyto- chrome, because of the single peak at 540 rim. In the biflagellate green alga Spermatozopsis similis, Kreimer et al. (1991) reported a 540 nm shoulder in the absortion spectrum of eyespot fractions, which


might be related to the as yet unidentified visual pigment for phototaxis in this alga. A rhodopsin was proposed to be the photoreceptor for the photomovements of the cell in Chlamydomonas reinhardtii (Foster et al. 1984). The absorption maximum of the "rhodopsin" in purified membranes of C. reinhardtii was 495 nm (Hegemann et al. 1991). The report by Deininger et al. (1995) of cloning and sequencing the "rhodopsin" in C. reinhardtii has led to the establishment of "rhodopsin(s)" as the respon- sible photoreceptor(s) (Hegemann et al. 1991, Law- son et al. 1991, Takahashi et al. 1991, Kreimer 1994), however, green-light receptor(s) in organisms other than C. reinhardtii and Spermatozopsis similis is/are still unknown. Reports concerning the green-light effect on chloroplast movements are scanty. Reorientation of chloroplast in Mougeotia sp. is induced by green-light, after the induction by intense blue- and near ultraviolet- light pulses (Gabry's 1985), if the electric vector of the green-light is vibrating perpendicularly to the lon- gitudinal cell axis (Lechowski et al. 1987). It is concluded from the experiments in this report that a green-light receptor, a 540 nm-absorbing pigment, regulates chloroplast dispersion by low light energy supplied for long duration and that a blue-light receptor, a 450nm-absorbing pigment, regulates chloroplast assemblage by high energy given for short durations. The action spectrum for the chloroplast assemblage in P. laevis displayed a pronounced peak at 450 nm. In Mougeotia sp., the effectiveness of blue light to induce chloroplast orientation was highest at 450 nm (Gabry's 1985). In Vaucheria sessilis (Fischer-Arnold 1963), Lemna trisulca (Zurzycki 1962), and Vallisne- ria spiralis (Seitz 1967), the action spectrum for chloroplast orientation had a peak at 450 nm with an auxiliary peak at 360 nm. The action spectra for phototaxis in zoospores in the brown alga Pseudo- chorda gracilis was dependent on the short wavelength with the two peaks at 420 and 450 nm (Kawai and Watanabe 1991). Flavin pigments have been the most likely candidate for the photoreceptor of the blue light (Zurzycki 1980, Jenkins et al. 1995, Mat- sunaga et al. 1997). Three peaks of absorption spectrum for riboflavin have been reported to be 450 nm, 375 nm, and 260 nm (Warburg and Christian 1938). Although in P. laevis the UV-A peak was not found at 375 nm but at 350 nm, the light receptor for the chloroplast assemblage in P. laevis also seems most probably to be a flavin pigment.

219

Phytochrome is widely involved in chloroplast orientation in higher plants and algae (Haupt 1965, Takagi et al. 1990, Watanabe 1995). In the chloroplast orientation of Mougeotia sp., far-red light can abolish chloroplast orientation induced by red light. Howev- er, in addition to phytochrome, continuous weak blue light (449 rim) can induce chloroplast orientation in the presence of a strong back ground of diffused far- red light, suggesting the existence of a different sensor pigment for blue light ("cryptochrome") (Gabry's et al. 1984). In Mesotaenium caldariorum, however, the phytochrome effect is strongly potentiated by blue light, although blue light itself is almost ineffective as the stimulus to bring about chloroplast orientation (Haupt 1966, Kraml et al. 1988). However, in P. laevis, broad-band red light (50% maximum fluence rate at 600 and 700 nm) of about 200 ~mol/m 2 �9 s did not induce chloroplast dispersion nor assemblage (data not shown). Therefore, phytochrome does not appear to directly regulate chloroplast movement. As for ecological significance of the photoregulated chloroplast movement, it is noteworthy that P. laevis grows at the muddy stone surface in gentle flow, gen- erally receiving an indirect sunlight. But sometimes when the cells receive sudden and direct irradiations of bright sunlight by physical conditions of the flow, all of the chloroplasts would begin to migrate to the central region of the cell so as to shade one another to protect themselves from the intense light. However, the meaning of the transiency of chloroplast assemblage is unknown.

Acknowledgements The authors wish to sincerely thank Mr. M. Kubota (National Insti- tute for Basic Biology) for help and much advice about experiments using the Okazaki Large Spectrograph; Dr. I. Ikegami and Dr. A. Kamiya (Teikyou University) for their invaluable advice about absorption spectra; Dr. C. Bowler (Stazione Zoologica, Naples) for critical reading of the manuscript. This work was done under the NIBB cooperative research program for the use of the Okazaki Large Spectrograph (nrs. 94-505, 95-513, 96-509).

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Green- and blue-light-mediated chloroplast migration in the centric diatomPleurosira laevis