Planta
Planta (1981) 152:553-556
9 Springer-Verlag 1~)81
Chloroplast translocations induced by light pulses Effects of single light pulses H. G a b r y g l , T. W a l c z a k
2,
a n d J. Z u r z y c k i l
1 Department of Plant Physiology, Institute of Molecular Biology, Jagellonian University, Grodzka 53, PL-31-001 Krakdw, and 2 Department of Nuclear Spectroscopy, Institute of Nuclear Physics, Radzikowskiego 152, PL-31-142 Krak6w, Poland
Abstract. The effect o f single blue-light pulses on c h l o r o p l a s t r e a r r a n g e m e n t was s t u d i e d in the leaves o f Tradescantia albiflora, Chlorophytum elatum, a n d Lemna trisulca. F o r m e a s u r i n g t r a n s l o c a t i o n s in terrestrial p l a n t s the m e t h o d o f t r a n s m i s s i o n changes was u s e d ; t r a n s l o c a t i o n s in the w a t e r p l a n t Lemna were s t u d i e d b y direct m i c r o s c o p i c o b s e r v a t i o n a n d counting. S t r o n g light (30 W m - 2) a p p l i e d in the f o r m o f s h o r t pulses, s h o r t e r t h a n a lag p e r i o d o f t r a n s l o c a tions, induces s o m e t r a n s i e n t effects in the following d a r k period. W i t h s h o r t pulses, t r a n s i e n t r e a r r a n g e m e n t s o f c h l o r o p l a s t s to a w e a k - l i g h t p o s i t i o n were found. W i t h l o n g e r pulse d u r a t i o n , b i p h a s i c responses t o o k place in Tradescantia a n d Lemna: The initial m o v e m e n t to a p a r t i a l s t r o n g - l i g h t p o s i t i o n was foll o w e d by a wave o f t r a n s l o c a t i o n to a w e a k - l i g h t a r r a n g e m e n t . In Chlorophytum this t y p e o f r e s p o n s e a p p e a r e d o n l y in a n a r r o w fluence range. T h e validity o f the r e c i p r o c i t y law in r e l a t i o n to fluence rate a n d time o f i r r a d i a t i o n was c o n f i r m e d for Tradescantia. T h e results m a y give us an insight into the kinetics o f the p r i m a r y effects o f light in the t r a n s l o c a t i o n process. K e y words: Blue light - C h l o r o p l a s t m o v e m e n t s Lemna - Light, c h l o r o p l a s t m o v e m e n t - Tradescantia.
Introduction In the p h y t o c h r o m e - m e d i a t e d c h r o m a t o p h o r e m o v e m e n t s in Mougeotia, a single light pulse evokes a stable g r a d i e n t o f the active f o r m o f p h y t o c h r o m e a l o n g the cell b o r d e r which induces c h r o m a t o p h o r e m o v e m e n t , even in the s u b s e q u e n t d a r k p e r i o d ( H a u p t 1959, 1960, 1980). In c o n t r a s t , for the blue l i g h t - m e d i a t e d t r a n s l o c a t i o n o f c h l o r o p l a s t s , a continu o u s i l l u m i n a t i o n was a s s u m e d to be necessary for
the i n d u c t i o n o f m o v e m e n t s . This a s s u m p t i o n was b a s e d on the fact that in d a r k - a d a p t e d cells, after a s h o r t light pulse, no a r r a n g e m e n t o f c h l o r o p l a s t s o t h e r t h a n the d a r k p o s i t i o n was o b s e r v e d as the end effect. H o w e v e r , the m e t h o d s o f r e g i s t r a t i o n o f c h l o r o p l a s t p o s i t i o n used so far were n o t very suitable for m e a s u r i n g t r a n s i e n t effects f o l l o w i n g a light pulse. A new m e t h o d d e v e l o p e d in this l a b o r a t o r y ( W a l c z a k a n d G a b r y g 1980) m a k e s p o s s i b l e the c o n t i n u o u s rec o r d i n g o f c h l o r o p l a s t position. F i r s t e x p e r i m e n t s , in which single light pulses were used, s h o w e d that in the p e r i o d o f 15-30 m i n f o l l o w i n g flash i r r a d i a t i o n s o m e r e a r r a n g e m e n t s t a k e place before the c h l o r o plasts r e t u r n to the stable d a r k position. This study was u n d e r t a k e n to c h a r a c t e r i z e these t r a n s i e n t effects a n d to o b t a i n s o m e i n f o r m a t i o n on the kinetics o f p r i m a r y effects o f light in the t r a n s l o c a t i o n processes.
Material and methods The detached leaves of two terrestrial plants, Tradescantia albijlora and Chlorophytum elatum, were used for the experiments. The anatomy of the leaves and the relations between chloroplast arrangements and light transmission in these species were described elsewhere (Oabry~ and Walczak 1980). For a comparison, the leaves (fronds) of a water plant, Lemna trisculca, a well known object for chloroplast translocation studies (Zurzycki 1962), were used. In all experiments, the leaves were pre-adapted in darkness for 12 h. Recordings of light transmission through the leaves as a criterion of chloroplast arrangement were performed on a double-beam photometer (Walczak and Gabry~ 1980). In order to obtain single light pulses, an electronically controlled shutter was introduced into the path of the actinic light. The opening and closing time of the shutter was about 10 ms. The duration of light pulses ranged from 20 ms to 100 s. Blue light selected from a halogen I00 W, 12 V radiation by glass filters BG12/2, BG23/2 and GGI3/2 mm had the maximum at 450 nm and a half-band width of 80 nm. Principally a fluence rate of 30 W m-2 was used, only in some cases was it reduced by a set of neutral filters. The effects of short blue light pulses on Lemna cells were studied microscopically by the counting method (Zurzycki 1962). The number of measured cells was reduced to 6 in order to repeat
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Fig. 1. Recordings of transmission changes in a Tradescantia leaf induced by continuous weak light (50 mW m -2) and strong light (19 W m 2) left, and by single light pulses (30 W m -2) right. The numbers by the arrows show the pulse duration in seconds. Starting position dark arrangement of chloroplasts. The corresponding transmission level denoted by dashed lines. Dark intervals between consecutive irradiations - 3 h
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Fig. 2a, b. The amplitudes of the positive and negative chloroplast response in a Tradescantia leaf in dependence on the pulse duration. The amplitudes calculated in relation to maximal responses induced by continuous light (a). The time measured from the beginning of light pulses to the appearance of the maximal positive and negative response (b). Means fl'om 4 experiments
Blue light o f a fluence rate of 30 W m - 2 acting continuously on a dark-adapted Tradescantia leaf induces a translocation of chloroplasts to a complete stronglight position. This translocation is connected with a significant increase in light transmission. The light of the same intensity, but used in the form of short pulses (from 20 ms to 1 s), induces transient decreases in light transmission (Fig. 1). Their maxima are attained after about 15 rain and amount to from about 10 to 60 percent of the changes characteristic of the complete weak-light rearrangement. Light pulses of longer duration (from 3 to 100 s) induce biphasic responses of light transmission: first an increase connected with a partial strong-light rearrangement followed by a wave of decrease characteristic of the weak-light rearrangement. 1 The mean maxima of the changes in dependence on the duration of the light pulse are presented in Fig. 2a. It is shown that at the used light intensity, a pulse shorter than 20 ms has a very small effect. At 300 ms the positive wave becomes nearly saturated. F r o m 3 s of light duration a negative wave appears, the magnitude of which increases with the time of irradiance, becoming saturated between 30 and 100 s. Pulses longer than 100 s were not used because we tried to keep the illumination within the lag phase of rearrangements. The bigger the negative wave, the more the maximum of the following positive response is reduced. Figure 2b presents the time necessary to obtain the maxima of positive and negative waves as measured from the beginning of pulse irradiation. T h e positive peak appears about 15 min after the application of a short light pulse (20 to 300 ms) and this value is nearly indepefident of the flash duration with a slight tendency to increase. Above 300 ms, the time necessary to obtain the positive maximum increases linearly with the logarithm of pulse duration. A negative response is always faster than a positive one, and the time of reaching its maximum increases linearly with the logarithm of the irradiation period, from 5 to 12 min. For checking the validity of the reciprocity law, a sequence of pulses 0.1, 1, 10 and 100 s was used. The fluence rate was 30 W m - 2 for the 0.1 s pulse and was reduced by a factor of 10 at every subsequent 1 Since both types of responses were induced with strong light, they will be denoted as positive (weak-light response type) and negative (strong light response type)
H. Gabry~ et al. : Chloroplast translocations induced by light
555
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Fig. 3. Chloroplast response in a Tradescantia leaf in the range of half saturation of positive reaction on a constant energy flux of 3 J 11-1-2applied in the form of pulses from 0.1 to 100 s duration. Lower curve - the maximum positive response induced by 1 s, 30 W m - 2 fluence rate
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Fig. 4. Chloroplast translocation in Lemna trisulca induced in the dark-adapted cells by single light pulses of 0.8 s (a) and 8 s (b) measured by the method of counting. The results are means from 6 cells. For both pulses, upper curves show the total number of chloroplasts in a flat position, i.e. situated at the cell wails perpendicular to the light direction. Lower curves indicate the number of chloroplasts situated at the cell wall turned towards the light as percent of alI chloroplasts in the flat position
longer pulse with a set of neutral filters. The energy fluence was 3 J m - 2 in every case. The results (Fig. 3) show that the magnitude and kinetics of chloroplast rearrangements are nearly the same in spite of the reduction of light intensity by 4 orders of magnitude. A Tradescantia leaf is composed of 2 to 3 layers of mesophyll cells. It might be supposed that the
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Chlorophytum chloroplasts on the single light as in Fig. i. The initial small rise in transmis100 s irradiations results most probably from changes of chloroplasts and not from their
recorded effects are the result of a superposition of different cell layers obtaining different light doses. To check this possibility the effects of light pulses on Lemna leaves were studied using the part of the lamina which only consists of a single layer of mesophyll cells. The results (Fig. 4a and b) show that in such an object positive or biphasic transient effects also exist, depending on the duration of 30 W m -2 blue light pulses. Counting of chloroplasts on the upper and lower cell walls indicated that the percentage of chloroplasts situated flatwise at the both walls is equal and principally constant (as is the case with the dark arrangement); only at the very beginning of translocation do the chloroplasts react faster at the wall turned away from light. The response of Lemna on single light pulses is very similar to that found for Tradescantia leaves. An example of diversity of the response by keeping the same general pattern are the reactions in Chlorophytum leaves (Fig. 5). The positive responses for short irradiations are slower - the mean time necessary to obtain the maximum is of the order of 25 min in comparison to 15 min in Tradescantia. The amplitude of maximal positive response increases with the pulse fluence, reaches a maximum, and decreases before the negative wave appears. By further increase of pulse duration only the negative wave is observed, its amplitude obtaining a maximal value and then decreasing. The return to the dark level is extremely slow and the subsequent positive wave does not appear (not shown in Fig. 5). The time necessary to get the maximal negative wave is nearly the same as that for Tradescantia.
556 Discussion
The arrangement of chloroplasts is controlled by light. It can be assumed that between the physical act of photoreceptor excitation and the end effect - translocation of chloroplasts - there must exist a chain of reactions showing their specific kinetics, where each successive step depends on the previous one (Zurzycki 1972). The first steps of this chain of processes may be denoted as X, Y etc. P* ~ X - - , Y .... ~ T r a n s l o c a t i o n The level of the state X created by light absorbed in photoreceptor is unstable because after switching off the illumination chloroplasts assume the dark position. This feature suggests that at a continuous irradiation the X state level results from a balance between a generation process having a rate constant kl (depending on light intensity) and a degradation process running with a rate constant k2
Xo/'', '"/"?X /,2 where n hv is the fluence rate. If the coefficient k2 is assumed to be constant, the level of X depends on the radiation density, being 0 in darkness (which corresponds to the dark position of chloroplasts), low at a tow light intensity (which evokes a weak-light position) and high in intense light (which corresponds to a specific strong-light position of chloroplasts). After switching off the strong light, the level of X (constant during irradiation) decreases with the rate constant k2. If the kinetics of this decrease is of the same order of magnitude as the kinetics of chloroplast translocation, one should expect the appearance of transient effects in the arrangement of chloroplasts. They should show the tendency to assume a weaklight arrangement when X passes through middle and low levels. This is really the case. It has been demonstrated in Lemna cells that the transfer from stronglight arrangement to dark arrangement proceeds through, at least partially, the weak-light arrangement, the m a x i m u m of which is obtained after 15 to 30 rain (cf. Zurzycka and Zurzycki 1953, Figs. 3, 4; Zurzycki 1962, Fig. 17). The effects of light pulses reported in this paper may be explained in a similar way. By the short light flash the generation of a certain level of the state X takes place. This level, by short light pulses, depends not only on the intensity
H. Gabryfiet al. : Chloroplast translocations induced by light but on the product of the intensity and the pulse duration, i.e., on the pulse fluence. In the subsequent dark period the level of X slowly decreases. The halflife time of this decrease is of the order of minutes. This may be concluded f r o m two facts: 1) the validity of the reciprocity law in the range from 0.1 to 100 s, and 2) the existence of translocations for more than 30 min after the light pulse. The chloroplasts react on the temporary level of X by their positive or negative response. The above explanation, treating the whole cell as a unit, is very simplified. In reality one must distinguish the events taking place at the best irradiated and the weakest irradiated cell walls, because, as shown in m a n y studies, the existence of an irradiation gradient is necessary to induce chloroplast rearrangements. The attempt to construct a physical model of these processes will be the topic of the next paper. We also hope that an explanation of a different behavior of Chlorophytum chloroplasts will be possible on the basis of this model. The physical nature of the state X is unknown. It may be a specific substance produced locally near the cell border or a specific state of the cell membrane. The second supposition seems more plausible. References
Gabryg H., Walczak T. (1980) Photometric study of chloroplast phototranslocations in leaves of 1and plants. Acta Physiol. Plant. 4, 281-290 Haupt W. (1959) Die Chloroplastendrehung bei Mougeotia I. (Jber den quantitativen und qualitativen Lichtbedarf der Schwachlichtbewegung. Planta 53, 484-501 Haupt W. (1960) Die Chloroplastendrehung bei Mougeotia II. Die Induktion der Schwachlichtbewegung durch linear polarisiertes Licht. Planta 55, 465-479 Haupt W., Hupfer B., Kramt M (1980) Blitzlichtinduktion der Chloroplastenbewegung bei Mougeotia. Wirkung unterschiedlicher Spektralbereiche und Polarisationsrichtungen. Z. Pflanzenphysiol. 96, 331 342 Walczak T, Gabryg H. (1980) New Type of photometer for measurements of transmission changes corresponding to chloroplast movements in leaves. Photosynthetica 14, 65-72 Zurzycka A., Zurzycki J (1953) Studies on the phototactic movements of chloroplasts I. Acta Soc. Bot. Polon. 22, 667 679 Zurzycki J (1962) The action spectrum for the light dependent movements of chloroplasts in Lemna trisulca L. Acta Soc. Bot. Polon. 31,489-538 Zurzycki J (1972) Primary reactions in the chloroplast rearrangements. Acta Protozool. 11, 189-199 Received 2 March; accepted 13 May 1981
Planta (1981) 152:553-556
9 Springer-Verlag 1~)81
Chloroplast translocations induced by light pulses Effects of single light pulses H. G a b r y g l , T. W a l c z a k
2,
a n d J. Z u r z y c k i l
1 Department of Plant Physiology, Institute of Molecular Biology, Jagellonian University, Grodzka 53, PL-31-001 Krakdw, and 2 Department of Nuclear Spectroscopy, Institute of Nuclear Physics, Radzikowskiego 152, PL-31-142 Krak6w, Poland
Abstract. The effect o f single blue-light pulses on c h l o r o p l a s t r e a r r a n g e m e n t was s t u d i e d in the leaves o f Tradescantia albiflora, Chlorophytum elatum, a n d Lemna trisulca. F o r m e a s u r i n g t r a n s l o c a t i o n s in terrestrial p l a n t s the m e t h o d o f t r a n s m i s s i o n changes was u s e d ; t r a n s l o c a t i o n s in the w a t e r p l a n t Lemna were s t u d i e d b y direct m i c r o s c o p i c o b s e r v a t i o n a n d counting. S t r o n g light (30 W m - 2) a p p l i e d in the f o r m o f s h o r t pulses, s h o r t e r t h a n a lag p e r i o d o f t r a n s l o c a tions, induces s o m e t r a n s i e n t effects in the following d a r k period. W i t h s h o r t pulses, t r a n s i e n t r e a r r a n g e m e n t s o f c h l o r o p l a s t s to a w e a k - l i g h t p o s i t i o n were found. W i t h l o n g e r pulse d u r a t i o n , b i p h a s i c responses t o o k place in Tradescantia a n d Lemna: The initial m o v e m e n t to a p a r t i a l s t r o n g - l i g h t p o s i t i o n was foll o w e d by a wave o f t r a n s l o c a t i o n to a w e a k - l i g h t a r r a n g e m e n t . In Chlorophytum this t y p e o f r e s p o n s e a p p e a r e d o n l y in a n a r r o w fluence range. T h e validity o f the r e c i p r o c i t y law in r e l a t i o n to fluence rate a n d time o f i r r a d i a t i o n was c o n f i r m e d for Tradescantia. T h e results m a y give us an insight into the kinetics o f the p r i m a r y effects o f light in the t r a n s l o c a t i o n process. K e y words: Blue light - C h l o r o p l a s t m o v e m e n t s Lemna - Light, c h l o r o p l a s t m o v e m e n t - Tradescantia.
Introduction In the p h y t o c h r o m e - m e d i a t e d c h r o m a t o p h o r e m o v e m e n t s in Mougeotia, a single light pulse evokes a stable g r a d i e n t o f the active f o r m o f p h y t o c h r o m e a l o n g the cell b o r d e r which induces c h r o m a t o p h o r e m o v e m e n t , even in the s u b s e q u e n t d a r k p e r i o d ( H a u p t 1959, 1960, 1980). In c o n t r a s t , for the blue l i g h t - m e d i a t e d t r a n s l o c a t i o n o f c h l o r o p l a s t s , a continu o u s i l l u m i n a t i o n was a s s u m e d to be necessary for
the i n d u c t i o n o f m o v e m e n t s . This a s s u m p t i o n was b a s e d on the fact that in d a r k - a d a p t e d cells, after a s h o r t light pulse, no a r r a n g e m e n t o f c h l o r o p l a s t s o t h e r t h a n the d a r k p o s i t i o n was o b s e r v e d as the end effect. H o w e v e r , the m e t h o d s o f r e g i s t r a t i o n o f c h l o r o p l a s t p o s i t i o n used so far were n o t very suitable for m e a s u r i n g t r a n s i e n t effects f o l l o w i n g a light pulse. A new m e t h o d d e v e l o p e d in this l a b o r a t o r y ( W a l c z a k a n d G a b r y g 1980) m a k e s p o s s i b l e the c o n t i n u o u s rec o r d i n g o f c h l o r o p l a s t position. F i r s t e x p e r i m e n t s , in which single light pulses were used, s h o w e d that in the p e r i o d o f 15-30 m i n f o l l o w i n g flash i r r a d i a t i o n s o m e r e a r r a n g e m e n t s t a k e place before the c h l o r o plasts r e t u r n to the stable d a r k position. This study was u n d e r t a k e n to c h a r a c t e r i z e these t r a n s i e n t effects a n d to o b t a i n s o m e i n f o r m a t i o n on the kinetics o f p r i m a r y effects o f light in the t r a n s l o c a t i o n processes.
Material and methods The detached leaves of two terrestrial plants, Tradescantia albijlora and Chlorophytum elatum, were used for the experiments. The anatomy of the leaves and the relations between chloroplast arrangements and light transmission in these species were described elsewhere (Oabry~ and Walczak 1980). For a comparison, the leaves (fronds) of a water plant, Lemna trisculca, a well known object for chloroplast translocation studies (Zurzycki 1962), were used. In all experiments, the leaves were pre-adapted in darkness for 12 h. Recordings of light transmission through the leaves as a criterion of chloroplast arrangement were performed on a double-beam photometer (Walczak and Gabry~ 1980). In order to obtain single light pulses, an electronically controlled shutter was introduced into the path of the actinic light. The opening and closing time of the shutter was about 10 ms. The duration of light pulses ranged from 20 ms to 100 s. Blue light selected from a halogen I00 W, 12 V radiation by glass filters BG12/2, BG23/2 and GGI3/2 mm had the maximum at 450 nm and a half-band width of 80 nm. Principally a fluence rate of 30 W m-2 was used, only in some cases was it reduced by a set of neutral filters. The effects of short blue light pulses on Lemna cells were studied microscopically by the counting method (Zurzycki 1962). The number of measured cells was reduced to 6 in order to repeat
0032-0935/81/0152/0553/$01.00
554
H. Gabry~ et al. : Chloroplast translocations induced by light 10 rain
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the registering as frequently as every 2 min. AI1 experiments were carried out at 20 ~ C.
I0,5%
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0.1
Results
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.
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.
.
.
.
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.
.
.
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.
.
.
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Fig. 1. Recordings of transmission changes in a Tradescantia leaf induced by continuous weak light (50 mW m -2) and strong light (19 W m 2) left, and by single light pulses (30 W m -2) right. The numbers by the arrows show the pulse duration in seconds. Starting position dark arrangement of chloroplasts. The corresponding transmission level denoted by dashed lines. Dark intervals between consecutive irradiations - 3 h
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Fig. 2a, b. The amplitudes of the positive and negative chloroplast response in a Tradescantia leaf in dependence on the pulse duration. The amplitudes calculated in relation to maximal responses induced by continuous light (a). The time measured from the beginning of light pulses to the appearance of the maximal positive and negative response (b). Means fl'om 4 experiments
Blue light o f a fluence rate of 30 W m - 2 acting continuously on a dark-adapted Tradescantia leaf induces a translocation of chloroplasts to a complete stronglight position. This translocation is connected with a significant increase in light transmission. The light of the same intensity, but used in the form of short pulses (from 20 ms to 1 s), induces transient decreases in light transmission (Fig. 1). Their maxima are attained after about 15 rain and amount to from about 10 to 60 percent of the changes characteristic of the complete weak-light rearrangement. Light pulses of longer duration (from 3 to 100 s) induce biphasic responses of light transmission: first an increase connected with a partial strong-light rearrangement followed by a wave of decrease characteristic of the weak-light rearrangement. 1 The mean maxima of the changes in dependence on the duration of the light pulse are presented in Fig. 2a. It is shown that at the used light intensity, a pulse shorter than 20 ms has a very small effect. At 300 ms the positive wave becomes nearly saturated. F r o m 3 s of light duration a negative wave appears, the magnitude of which increases with the time of irradiance, becoming saturated between 30 and 100 s. Pulses longer than 100 s were not used because we tried to keep the illumination within the lag phase of rearrangements. The bigger the negative wave, the more the maximum of the following positive response is reduced. Figure 2b presents the time necessary to obtain the maxima of positive and negative waves as measured from the beginning of pulse irradiation. T h e positive peak appears about 15 min after the application of a short light pulse (20 to 300 ms) and this value is nearly indepefident of the flash duration with a slight tendency to increase. Above 300 ms, the time necessary to obtain the positive maximum increases linearly with the logarithm of pulse duration. A negative response is always faster than a positive one, and the time of reaching its maximum increases linearly with the logarithm of the irradiation period, from 5 to 12 min. For checking the validity of the reciprocity law, a sequence of pulses 0.1, 1, 10 and 100 s was used. The fluence rate was 30 W m - 2 for the 0.1 s pulse and was reduced by a factor of 10 at every subsequent 1 Since both types of responses were induced with strong light, they will be denoted as positive (weak-light response type) and negative (strong light response type)
H. Gabry~ et al. : Chloroplast translocations induced by light
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Fig. 4. Chloroplast translocation in Lemna trisulca induced in the dark-adapted cells by single light pulses of 0.8 s (a) and 8 s (b) measured by the method of counting. The results are means from 6 cells. For both pulses, upper curves show the total number of chloroplasts in a flat position, i.e. situated at the cell wails perpendicular to the light direction. Lower curves indicate the number of chloroplasts situated at the cell wall turned towards the light as percent of alI chloroplasts in the flat position
longer pulse with a set of neutral filters. The energy fluence was 3 J m - 2 in every case. The results (Fig. 3) show that the magnitude and kinetics of chloroplast rearrangements are nearly the same in spite of the reduction of light intensity by 4 orders of magnitude. A Tradescantia leaf is composed of 2 to 3 layers of mesophyll cells. It might be supposed that the
...........
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Fig. S. Response of pulses. Explanation sion seen at 30 and the conformational translocations
,00
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Chlorophytum chloroplasts on the single light as in Fig. i. The initial small rise in transmis100 s irradiations results most probably from changes of chloroplasts and not from their
recorded effects are the result of a superposition of different cell layers obtaining different light doses. To check this possibility the effects of light pulses on Lemna leaves were studied using the part of the lamina which only consists of a single layer of mesophyll cells. The results (Fig. 4a and b) show that in such an object positive or biphasic transient effects also exist, depending on the duration of 30 W m -2 blue light pulses. Counting of chloroplasts on the upper and lower cell walls indicated that the percentage of chloroplasts situated flatwise at the both walls is equal and principally constant (as is the case with the dark arrangement); only at the very beginning of translocation do the chloroplasts react faster at the wall turned away from light. The response of Lemna on single light pulses is very similar to that found for Tradescantia leaves. An example of diversity of the response by keeping the same general pattern are the reactions in Chlorophytum leaves (Fig. 5). The positive responses for short irradiations are slower - the mean time necessary to obtain the maximum is of the order of 25 min in comparison to 15 min in Tradescantia. The amplitude of maximal positive response increases with the pulse fluence, reaches a maximum, and decreases before the negative wave appears. By further increase of pulse duration only the negative wave is observed, its amplitude obtaining a maximal value and then decreasing. The return to the dark level is extremely slow and the subsequent positive wave does not appear (not shown in Fig. 5). The time necessary to get the maximal negative wave is nearly the same as that for Tradescantia.
556 Discussion
The arrangement of chloroplasts is controlled by light. It can be assumed that between the physical act of photoreceptor excitation and the end effect - translocation of chloroplasts - there must exist a chain of reactions showing their specific kinetics, where each successive step depends on the previous one (Zurzycki 1972). The first steps of this chain of processes may be denoted as X, Y etc. P* ~ X - - , Y .... ~ T r a n s l o c a t i o n The level of the state X created by light absorbed in photoreceptor is unstable because after switching off the illumination chloroplasts assume the dark position. This feature suggests that at a continuous irradiation the X state level results from a balance between a generation process having a rate constant kl (depending on light intensity) and a degradation process running with a rate constant k2
Xo/'', '"/"?X /,2 where n hv is the fluence rate. If the coefficient k2 is assumed to be constant, the level of X depends on the radiation density, being 0 in darkness (which corresponds to the dark position of chloroplasts), low at a tow light intensity (which evokes a weak-light position) and high in intense light (which corresponds to a specific strong-light position of chloroplasts). After switching off the strong light, the level of X (constant during irradiation) decreases with the rate constant k2. If the kinetics of this decrease is of the same order of magnitude as the kinetics of chloroplast translocation, one should expect the appearance of transient effects in the arrangement of chloroplasts. They should show the tendency to assume a weaklight arrangement when X passes through middle and low levels. This is really the case. It has been demonstrated in Lemna cells that the transfer from stronglight arrangement to dark arrangement proceeds through, at least partially, the weak-light arrangement, the m a x i m u m of which is obtained after 15 to 30 rain (cf. Zurzycka and Zurzycki 1953, Figs. 3, 4; Zurzycki 1962, Fig. 17). The effects of light pulses reported in this paper may be explained in a similar way. By the short light flash the generation of a certain level of the state X takes place. This level, by short light pulses, depends not only on the intensity
H. Gabryfiet al. : Chloroplast translocations induced by light but on the product of the intensity and the pulse duration, i.e., on the pulse fluence. In the subsequent dark period the level of X slowly decreases. The halflife time of this decrease is of the order of minutes. This may be concluded f r o m two facts: 1) the validity of the reciprocity law in the range from 0.1 to 100 s, and 2) the existence of translocations for more than 30 min after the light pulse. The chloroplasts react on the temporary level of X by their positive or negative response. The above explanation, treating the whole cell as a unit, is very simplified. In reality one must distinguish the events taking place at the best irradiated and the weakest irradiated cell walls, because, as shown in m a n y studies, the existence of an irradiation gradient is necessary to induce chloroplast rearrangements. The attempt to construct a physical model of these processes will be the topic of the next paper. We also hope that an explanation of a different behavior of Chlorophytum chloroplasts will be possible on the basis of this model. The physical nature of the state X is unknown. It may be a specific substance produced locally near the cell border or a specific state of the cell membrane. The second supposition seems more plausible. References
Gabryg H., Walczak T. (1980) Photometric study of chloroplast phototranslocations in leaves of 1and plants. Acta Physiol. Plant. 4, 281-290 Haupt W. (1959) Die Chloroplastendrehung bei Mougeotia I. (Jber den quantitativen und qualitativen Lichtbedarf der Schwachlichtbewegung. Planta 53, 484-501 Haupt W. (1960) Die Chloroplastendrehung bei Mougeotia II. Die Induktion der Schwachlichtbewegung durch linear polarisiertes Licht. Planta 55, 465-479 Haupt W., Hupfer B., Kramt M (1980) Blitzlichtinduktion der Chloroplastenbewegung bei Mougeotia. Wirkung unterschiedlicher Spektralbereiche und Polarisationsrichtungen. Z. Pflanzenphysiol. 96, 331 342 Walczak T, Gabryg H. (1980) New Type of photometer for measurements of transmission changes corresponding to chloroplast movements in leaves. Photosynthetica 14, 65-72 Zurzycka A., Zurzycki J (1953) Studies on the phototactic movements of chloroplasts I. Acta Soc. Bot. Polon. 22, 667 679 Zurzycki J (1962) The action spectrum for the light dependent movements of chloroplasts in Lemna trisulca L. Acta Soc. Bot. Polon. 31,489-538 Zurzycki J (1972) Primary reactions in the chloroplast rearrangements. Acta Protozool. 11, 189-199 Received 2 March; accepted 13 May 1981
