the effects of blue and red light on acetabularia mediterranea after a long dark period: recovery of...

The effects of blue and red light on Acetabularia mediterranea after a long dark period: recovery of the endogenous rhythms of transcellular electrical potential and

chloroplast velocity

H ~ L ~ N E BORGHI, SIMONE PUISEUX-DAO, A N D ANNE-CATHERINE DAZY Laborrltoire de Cytophysiologie vtgttale et de Toxicologie cellulaire,

Unitt AssociLe Centre National de la Redzerche scienti'que, 567, Universitt Paris VII , 2 Place Jussieu, 75251 Paris CPdex 05, France

Received May 10, 1985

BORGHI, H., S. PUISEUX-DAO, and A.-C. DAZY. 1986. The effects of blue and red light on Acetabularia nlerlitcrranea after a long dark period: recovery of the endogenous rhythms of transcellular electrical potential and chloroplast velocity. Can. J. Bot. 64: 1134- 1137.

After a long dark period (2 -8 weeks), the transcellular electrical potential along the cell of Acetabularia mediterranea has almost disappeared and cytoplasmic streaming is at a stop. Irradiation with continuous blue light (450 nm) induces the transcellular electrical potential to increase and oscillate. After two shorter oscillations, the rhythm becomes firn~ly established with a period around 23 h. This behaviour is similar to that observed in white light, where the final period is around 26 h. White light and blue light induce the appearance of endogenous diurnal oscillations in the velocity of the cytoplasmic streaming which resumes following a periodicity pattern similar to that of the transcellular electrical potential. Inadiation with continuous red light (650 nm) causes a strong increase of transcellular electrical potential and recovery of cytoplasmic streaming but seldom induces these two processes to resume with the typical oscillations. However, after irradiation with blue light, the rhythm of transcellular electrical potential persists for some time in red light but disappears progressively. When blue light is given after red light, the transcellular electrical potential rhythm resumes immediately. Blue light may be considered as a "Zeitgeber."

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BORGHI, H., S. PUISEUX-DAO et A.-C. DAZY. 1986. The effects of blue and red light on Acetabularia mediterranea after a long dark period: recovery of the endogenous rhythms of transcellular electrical potential and chloroplast velocity. Can. J. Bot. 64: 1134-1 137.

Aprks un long sCjour B I'obscuritC (2 -8 semaines), le potentiel Clectrique transcellulaire le long de la cellule d'Acetabularia rnediterranea est pratiquement annul6 et les mouvements cytoplasmiques sont arr&tCs. L'illumination des algues par de la lumikre bleue (450 nm) continue provoque une augmentation du potentiel Clectrique transcellulaire dont la valeur oscille. Apks deux oscillations courtes, la pCriode s'ttablit et se maintient autour de 23 h. Ce comportement est analogue B celui observC en lumikre blanche avec laquelle cependant, la pCriode finale est supkrieure, de I'ordre de 26 h. La lumikre bleue et la lumikre blanche induisent aussi l'apparition d'un rythme endogkne de la vitesse de dkplacement des chloroplastes ~'Ctablissant avec la m&me succession de pCriodes croissantes. L'illumination par la lumikre rouge (650 nm) continue provoque une forte augmentation du potentiel Clectrique transcellulaire et la reprise des mouvements cytoplasmiques mais elle permet rarement d'observer ce m&me type d'oscillations. Le rythme du potentiel Clectrique transcellulaire induit par la lumikre bleue peut cependant persister pendant quelque temps en lumikre rouge mais disparaft progressivement. Aprks un Cclairement en lumikre rouge, la lumikre bleue induit immkdiatement la reprise du rythme du potentiel Clectrique tr-nscellulaire. La lumikre bleue peut &tre considCrCe comme un <<Zeitgebe~>.

Introduction In Acetabularia, many parameters are subject to diurnal os-

cillations and many of these rhythms have been characterized as circadian (for reviews, see Schweiger and Schweiger 1977; Van den Driessche 1980; Puiseux-Dao 1984). Two of these circadian rhythms, photosynthetic oxygen evolution (Mergen- hagen and Schweiger 1973; Karakashian and Schweiger 1976) and direction of chloroplast migration (Broda et al. 1979), have been followed over months under constant light.

The transcellular electrical potential (VAB = 5 - 10 mV) is also subject to diurnal oscillations (Novak and Sironval 1976; Dazy et al. 1980), which persist for months under constant illumination (Broda and Schweiger 1981). In individual cells

The wavelength of the light is of great importance: blue light is the only morphogenetic wavelength (e.g., see Schmid and Clauss 1977). In addition blue light is known to induce many cellular or physiological events in plants and microorganisms. It has been suggested that flavin and (or) carotenoid receptors localized at the level of the plasma membrane are responsible for all blue light induced phenomena or the "blue-light syndrome" (Senger 1979, 1982; Briggs and Iino 1983).

The results reported here describe the influence of red and blue light on the recovery of VAB rhythm after a period of darkness. We also observed the recovery of cytoplasmic streaming and its time coupling with the electrical rhythm.

- the period may change slowly during such long-term measurements (Broda and Schweiger 1981). Although this rhythm may Materials and methods

also persist up to 7 days when the light is switched off, it Cells of Acetabularia rnediterranea were cultivated in synthetic

damps down progressively in the dark as the transcellular elec- Seawater 'LMS" (She~hard 1970) at 20-220C. Algae cm

trical potential disappears (Borghi et 1983). When the light long without a cap were placed in the dark for 2-8 weeks. presenting a clear zone in the middle of the stalk (resulting from the is switched On after 2-8 weeks of dark the progressive accumulation of chloroplasts at both ends (see Borghi

cal polarity of the reappears and, even under 01. 1983)) were chosen both for microscopic observations and light, the usual rhythm is restored after two brief oscillations of electrical measurements. As it is very difficult to manipulate the algae 2-5 h for the first period (mom, in Fig. l a ) and in the green inactinic light, we chose the dim red light from a dark- 13- 16 h for the second (m,m2 in Fig. la) (Borghi et al. 1983). room red lamp (Osram). Irradiation with this light induces a very

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BORGHI ET AL. 1135

small electrical response by the algae, which disappears immediately on extinguishing the light. In the case of electrophysiological experiments, cells were kept at least 24 h in the dark after being placed in the cuvette. Illumination with red or blue light was canied out with the use of Balzers filtraflex interferential filters K65 (650 nm) and K45 (450 nm), respectively (energy range: 3-5 W . m-2).

The potential difference between the apex and the base of the cell (VAB = VaFx - Vb;,,e) was measured externally. For each registration, a group of five algae closely packed together was placed in a three- compartment cuvette; the electrical setup was as described by Novak and Bentrup (1972). "MS" agar bridges connected the compartments to Ag-AgCI electrodes in 3 M KCI. VAB was continuously recorded on a chart at a speed of 3 cm . h-' by a pen recorder with a lo6 S?, input impedance (Bryans). To monitor the circadian component of the potential difference, only the values of the steady-state potential (VAB) were plotted each hour, while spontaneous action potentials (Grad- mann 1976) were neglected.

For cytological observations, the algae were placed in a small cuvette on the stage of a Zeiss microscope equipped with Nomarski interference differential contrast. The whole algae were also filmed at low magnification ( x 0.5) and 1 frame . min-' on 16-mm Ilford Pan F film type 752 P. The rate of organelle transport was estimated using frame by frame analysis of the time-lapse films.

For statistical comparison of the periods of rhythms in blue, red, o r white light, we calculated the mean value for each separate experi- ment (3 to 18 periods in each case) and compared these means, using the nonparametric test of Mann and ~ h i t n e y (U-test), because of thk small representation in number of each sample (7 experiments in blue light; 2 cases with rhythm in red light among 7 experiments; 18 experiments in white light).

Results Transcellular potential, V,,

In our experimental conditions, after 2-8 weeks of darkness, continuous blue light (BL) induced an increase of VAB and its circadian oscillation, as did continuous white light (WL) (Figs. l a and lb). In WL, the rhythm (similar to that of algae which had never been kept in the dark) was restored after two oscillations with a short period (m, to m, and m, to m, on Fig. la) . In BL, the first period was 3-5 h and the second about 10 h (Fig. 3). The mean period of the endogenous rhythm was shorter in BL (22.5 h) than in WL (26 h) (accord- ing to the U-test of Mann and Whitney, the difference between the two values is significant at a level of 1 %). In the same conditions and at an equal energy level, continuous red light (RL) also induced an increase in VAB, which reached high sustained values (10 mV and even 25 mV in one case) but did not present oscillations of the order of 24 h in 5 of the 7 groups of algae tested during at least 48 h (Fig. 2). The other 2 groups gave, after two short oscillations, a rhythm (Fig. lc) with a short mean period (20 h, Fig. 3). The U-test indicates that the difference between the periods in BL and RL is significant at the level of 5 %.

Increasing the energy of BL to 70 - 80 W . m-2 only changed the mean value of VAB and the amplitude of the rhythm, which were both raised, but did not change the period of the rhythm. At the same energy level, RL did not induce circadian rhythm of VAB.

After BL to RL transition, VAB rhythm disappeared in 24 h in two cases but was maintained over 8 periods of about 20 h in a third.

Upon RL to BL transition, VAB fell abruptly but briefly (4- 12 min) to zero, after which the cell polarity was progressively restored and the rhythm returned without the progressive increase in the period length observed after dark to BL or WL transition (Fig. 2b).

I 1 -f-.-., -

o 10 5 o hours

light on

FIG. 1. VAB rhythm: induction by continuous illumination after dark treatments (D). (a) White light (WL); D, 7 weeks. ( b ) Blue light (BL), 450 nm; D, 2 weeks. (c) Red light (RL), 650 nm; D, 3 weeks. Energy range: 3-5 W . m-'. Fig. l c represents one of the two cases observed where RL induced recovery of the VAB rhythm.

Chloroplast velocity The recovery of cytoplasmic streaming in WL after a pro-

longed dark treatment has been previously described in detail (Borghi et al. 1983). Over a period of several weeks without light (2-8 weeks, depending on the culture batches), the chloroplasts tend to collect in the base and in the apex, leaving a "clear zone" between the two. There is no more streaming. When such algae are then subjected to light under the microscope, the resumption of cytoplasmic streaming and the movement of organelles into the clear area may be readily observed. These movements occur transiently for about 15 min of WL or BL and stop again. After more than 1.5 but less than 4 h of light, true streaming recovers. Time-lapse films at low magnification ( ~ 0 . 5 ; 1 frame. min-') show that when streaming becomes firmly established, the speed of chloroplast clusters which are clearly visible in the "clear zone" oscillates in continuous WL or BL and cytoplasmic streaming (as a whole) regularly comes to a full or almost full stop. Moreover, we also observed that the number of plastid clusters in the "clear zone" increases when the movement is fast and decreases when it slows down or stops (because of chloroplast accumulation mainly at the base of the cell). The times at which the velocity minima occurred in continuous WL were estimated directly from the projection of time-lapse films (Table 1). The speed of the chloroplast clusters in the "clear zone" was measured at velocity maxima and minima on 14 algae, using frame by frame analysis, and reported in Table 2. The velocity rhythm was also indirectly evaluated by counting the clusters

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1136 CAN. J . BOT. VOL. 64. 1986

0 10 5 0

1 l i g h t o n

h o u r s

0 10 50 roo h o u r s

l i g h t o n

FIG. 2. ViB rhythm: induction by continuous illumination after dark treatments (D). (a) Red light (RL); D, 3 weeks. (b) Red light (RL) followed by blue light (BL); D, 4 weeks. Most frequently RL did not induce VAB to oscillate (circadian component). The daily rhythm re- sumed immediately in BL.

TABLE 1. Comparison of the lengths of the first periods of VAB and chloroplast speed rhythms induced by continuous

illumination with white light (3 -5 W . m-=)

Chloroplast speed Period VAB rhythm rhythm

TI ( ~ 2 ~ ~ 2 ~ ) 2-5 1.5 -4 T3 (mlm2) 13-16 12-18 T5 ( I ~ ~ I I ~ ~ ) 23-29 33 -39

NOTE: Values are expressed in hours. See Fig. 3 Ihr the signilicance of T,. T,, T5 and nr,. rrr, , 111,. n l , .

or by automatically recording optical density of the time-lapse films in a limited area of the "clear zone" (Fig. 4).

The results show that in continuous WL the number of clusters in the "clear zone" oscillates with time as does chloroplast speed, with the same increase of the period length as for VAB rhythm. A correct estimation of the period of the reestab- lished diurnal rhythm is difficult because of the rapid "refilling" of the "clear zone" with organelles. In continuous WL, this refilling allows one to study the cytoplasmic rhythm clearly during 50-60 h (optical density in the "clear zone" reaches 75% of maximum in 10-20 h of WL). In continuous BL, the transporl of chloroplasts is so effective that the "clear zone" rapidly disappears (optical density in this area reaches 75% of maximum in 1 - 10 h of BL).

In continuous RL, streaming of thin cytoplasmic strands can be observed during the first 10 min, then the cytoplasmic

h o u r s

301

I t l m e

' l i g h t o n

FIG. 3. Recovery of VAB rhythm. This figure shows (i) increase of the period length (T) during the first hours of continuous illumination and (ii) fluctuations of period values when rhythms have been re- established. Each point is the mean obtained from 7 groups of five algae in BL, 2 groups in RL, and 18 groups in WL. The caption represents a schematic drawing of YiB oscillations from onset of continuous light. T, time interval between two minima o r two maxima and taken as period length value.

TABLE 2. Chloroplast speed during the first hours of continuous illumination with white light (3 -5 W . m 2 )

m I M2 ~ n z M3 m3

VF 0-0 .8 3 -8 0-2.5 1.5-5 0-0.8 VM 5 20 5 8 2 .5

NOTE: Speed was estimared from frarnc by frame analysis of time-lapse films ( I kame. min-I). Values are expressed in microrne~res per second. VF, range of more frequently recorded speeds of chloroplasrs clusren cin-u- lating in the clear zone, V,,, maximum values recorded. See Figs. 3 and 4 for the significance of ni,, n r , , 171,. and M,, M,.

movements stop and restart 2 or 3 h later, but no clear rhythm of the order of 24 h has been detected. The cluster velocity is rather constant (VF = 1.5 -5 pm . s-'; VM = 8 pm . S-'; same notation as in Table 2) and the clusters, less numerous than in WL, are easily visible in the "clear zone" during a long time (optical density reaches 75% of maximum after 55 h of RL).

Discussion 'The rhythms described here (VAB rhythm and chloroplast

speed rhythm) are clearly endogenous since only one nonoscil- lating signal of the environment (light on) is sufficient to induce them after they have been suppressed by a long dark treatment.

Since we have observed that low velocities correspond to phases of accumulation of plastids mainly in the base, the rhythm of chloroplast speed we have described here and that of

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BORGHI ET AL. 1137

FIG. 4. Chloroplast speed rhythm. The number of chloroplast clusters (N) in a reference area of the "clear zone" have been plotted versus time. High densities at M, M,, M3 correspond to high speeds and low densities at m,, m,, m3 to low speeds or even to transient arrests of cytoplasmic movements. D, 4 weeks.

the direction of chloroplast migration reported by Koop (1978) could be two aspects of the same rhythm.

BL is very effective in inducing VAB and chloroplast speed rhythms. The ability of continuous BL to restore the photosynthetic rhythm after photosynthesis has come to a stop in R L has been described by Clauss (1979). Schmid (1981) also showed that BL may change the phase of the rhythm of chloroplast migration. Our results then reinforce the idea that BL may be regarded as a "Zeitgeber" for Acetabularia mediterranea. R L on the contrary seems ineffective in restoring VAB and streaming rhythms, although VAB rhythm may be maintained in R L following several hours of BL irradiation, as may occur in darkness after W L (Borghi er al. 1983). In BL, as well as in RL, the period of VA, rhythm is shorter than in W L , as is the case for the rhythm of the direction of chloroplast migration (Schmid 1981). This could mean that events occumng in BL and R L operate in a cooperative way influencing the value of the period in WL.

In both VAB and chloroplast rhythms, the established oscillations are preceded by a similar "adaptation" phase in which the period increases progressively before reaching a circadian value. This suggests a link between the two rhythms. Varia- tions of pH o r K' concentration, for example, would affect both VAB and the gel-sol state of the cytoplasm. In addition, light-induced synthesis in the cytoplasm could affect the plasma membrane composition and its functioning. Simultan- eous recording of the two rhythms and of the O2 production rhythm by the chloroplasts, in red or blue light, as well as the use of inhibitors of protein synthesis o r photosynthesis would permit one to assess whether there are several independent but interacting oscillators o r only one major oscillator indirectly controlling most metabolic activities of the cell.

Acknowledgements W e thank Ms. Guillo for maintenance of the cultures, Ms.

M . J . Lapkgue for technical assistance, Ms. S . Djaoui for typing the manuscript, and Ms. J. Marie for verifying the English. The time-lapse films were made with the skillful technical assistance of Mr. J . M . Provost.

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the effects of blue and red light on acetabularia mediterranea after a long dark period: recovery of...

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