Pl anta

Plan ta (1991)185 : 397-400

9 Springer-Verlag1991

Induction of oscillations in chlorophyll fluorescence by re-illumination of intact isolated pea chloroplasts Sonja Veljovi~-Jovanovi~ 1.* and Zoran G. Cerovi~ 2.

1 INEP, Institute of Pesticides and Environmental Protection, P.O. Box 46, Banatska 31b, and 2 Institute of Botany, University of Belgrade, Takovska 43, YU-11000 Belgrade, Yugoslavia Received 16 January; accepted 3 May 1991

Abstract. Oscillations in chlorophyll fluorescence yield were observed upon re-illumination o f intact isolated pea (Pisum sativum L.) chloroplasts that had attained their maximal rate of photosynthesis and had spent a short period in darkness. The oscillations depended on the length of the previous dark period, the length of previous illumination, and the reaction temperature. This finding confirms the presence of an "oscillatory center" in the chloroplasts temselves.

Chlorophyll fluorescence (oscillations) Chloroplast (intact, isolated) - Photosynthesis (oscillations) - P i s u m (chlorophyll fluorescence oscillations)

Key words:

Introduction

The question of the origin of oscillations o f photosynthesis has been raised since they were first recorded (MeAlister and Myers 1940; Van der Veen 1949; Bannister 1965; Canvin 1978; Ogawa 1982; Walker et al. 1983a, b; Stitt 1986; Peterson et al. 1988; Stitt et al. 1988; Stitt and Schreiber 1988). It is now obvious that these oscillations are a manifestation of the overall photosynthetic process as they have been detected in oxygen evolution (Bannister 1965; Ogawa 1982; Walker et al. 1983a, b; Havaux 1988; Stitt et al. 1988; Stitt and Schreiber 1988), carbon-dioxide uptake (McAlister and Myers 1940; Van der Veen 1949; Canvin 1978; Ogawa 1982; Peterson et al. 1988), chlorophyll fluorescence (McAlister and Myers 1940; Ogawa 1982; Walker et al. 1983a, b; Sivak et al. 1985; Peterson et al. 1988; Stitt and Schreiber 1988), and changes in concentrations of nucleotides (Lewenstein and Bachofen 1972; Stitt 1986) and intermediates (Wilson and Calvin 1955; Stitt 1986; Stitt et al. 1988). Still, a controversy exists concerning the * To whom correspondence should be addressed ** Present address: Center for Multidisciplinary Studies of the Belgrade University, S. Penezica-Krcuna 35, YU-11000 Belgrade, Yugoslavia

"oscillatory center", i.e. the group of reactions which together directly generate oscillations, whose identification would help us to understand the overall regulation and limitations in photosynthesis. It has been proposed that the activity and regulation of the system for sucrose synthesis, located in the cytosol, generates oscillations through its effect on the rate of phosphate recycling to chloroplasts (Stitt 1986; Stitt et al. 1988; Stitt and Schreiber 1988). Although based on measured changes in fructose 2,6-bisphosphate levels, which are known to regulate the pathway for sucrose synthesis, and other metabolites during oscillations (Stitt et al. 1988), this proposal could not be confirmed by a mathematical model (Laisk et al. 1989). Simulations of oscillations in photosynthesis using other mathematical models (Giersch 1986; H o r t o n and Nicholson 1987; Laisk and Walker 1989) and experiments on protoplasts (Furbank and H o r t o n 1987) argued in favor of an "oscillatory center" inherent to chloroplasts. The obvious approach was therefore to try to induce oscillations in isolated chloroplasts. The results published upto now (Plesni~ar and Cerovit; Fejzo et al. 1986; Carver and H o r t o n 1987; N a k a m o t o et al. 1987) have not been sufficiently convincing, partly because of the artificiality of the conditions needed to induce the observed transients (or oscillations) in photosynthesis. In this communication we describe oscillations of chlorophyll fluorescence yield linked to oxygen evolution, induced in highly intact isolated chloroplasts by re-illumination after a short dark period. The occurrence of oscillations was dependent on the length of the dark period and the physiological state of the chloroplasts which varied with the time of illumination in vitro. These findings confirm the existence in the chloroplast of an "oscillatory center" which is independent of cytosolic reactions.

Material and methods

Intact chloroplasts were isolated mechanically from leaves of 12-13-d-old pea plants (Pisum sativum L., cv. Petit Provengal, Seme, Belgrade, Yugoslavia) according to the method of Cerovi6 and Plesnitar (1984). Pea plants were grown in water culture in a con-

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time (s) Fig. 1. Time course of carbon-dioxide-dependent oxygen evolution ( - - - ) , chlorophyll fluorescence ( - - ) , qe (D) and qs (A) in intact isolated pea chloroplasts in suspension. The reaction mixture contained 330 mM sorbitol, 10 mM KC1, 1 mM EDTA, 50 mM HepesKOH, pH 7.9, 3 mM NaHCO3, 1 mM ATP, 5 mM Na4P2Ov, 2000 units catalase, 20 lag chloroplast chlorophyll in the final volume of 1 ml. The experiment was carried out at 27 ~ C and actinic light was 2800 lamol photons, m - 2 . s - 1 on the surface of the light guide. Modulated fluorescence was measured by a PAM-101 fluorimeter as described in Material and methods

trolled-growth chamber: 11 h light (350 lamol photons - m-2 . S - 1) at 18~ C, and 13 h in darkness at 12~ C. Before chloroplast isolation, plants were kept in darkness for 22 h, and then illuminated with 700 lamol photons - m - 2. s- 1 for 40 rain. The intactness of isolated chloroplast was above 92% as measured by the ferricyanide assay (Walker et al. 1987). Oxygen evolution and chlorophyll fluorescence were measured simultaneously using a Clark-type oxygen electrode in a chamber for liquid samples (Hansatech, King's Lynn, Norfolk, UK) designed originally by Delieu and Walker (1983). The differential of the oxygen signal was produced using a passive RC network. The actinic light (2800 lamol photons 9m -z 9s- 1, on the surface of the light guide) was provided by a 250-W quartz-halogen lamp and filtered through a heat-absorbing glass and a blue glass filter (4-96; Corning, New York, USA). Measurements of modulated chlorophyll fluorescence were made using a pulse-amplitudemodulation fluorimeter (PAM-101 ; H. Waltz, Effeltrich, FRG). A saturating pulse of light (16000 ~tmol photons, m -2 9s - 1) lasting 1 s was flashed every 10 s to fully reduce QA (the "primary" acceptor of photosystem II). The intensity and length of light pulses were determined to be saturating under these conditions. Values of the redox-state-dependent chlorophyll fluorescence quenching (qp) and non-photochemical chlorophyll fluorescence quenching (qN) were calculated according to Schreiber et al. (1986). Experiments were carried out in a reaction mixture containing 330mM sorbitol, 10 mM KC1, 1 mM EDTA, 3 mM NaHCO3, 1 mM ATP, 5 mM Na,,PzOT, 2000 units catalase, 50mM 4-(2-hydroxyethyl)-lpiperazineethane-sulfonic acid (Hepes) buffer, adjusted to pH 7.9 with KOH and 20 lag chloroplast chlorophyll. The final volume was 1 ml, and the temperature 27~ C. Chlorophyll concentration was determined according to Bruinsma (1961). Catalase (EC 1.11.1.6.) was purchased from Sigma (St. Louis, Mo., USA) and other reagents were of proanalysis grade.

Results and discussion W e choose to m e a s u r e c h l o r o p h y l l fluorescence in parallel with oxygen e v o l u t i o n because it has been d e m o n -

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Fig. 2. Induction of oscillations in chlorophyll fluorescence by re-illuminationof intact pea chloroplasts in suspension (conditions as in Fig. 1). When the maximum rate of oxygen evolution was reached (after 5 rain of illumination)chloroplasts were darkened for 20 s and then re-illuminated for 90 s (trace 2). The 20-s-dark/90-slight sequence was continued and the changes in chlorophyll fluorescence during the light periods are shown in traces 3 to 7

strated to be closely related to either oxygen e v o l u t i o n or c a r b o n - d i o x i d e fixation, with the a d v a n t a g e o f h a v i n g a m o r e favorable signal-to-noise ratio (McAlister a n d Myers 1940; O g a w a 1982; W a l k e r et al. 1983a, b ; Stitt a n d Schreiber 1988). I n a d d i t i o n , using the p u l s e - s a t u r a tion technique (Schreiber et al. 1986) we could characterize different c o m p o n e n t s o f chlorophyll fluorescence q u e n c h i n g a n d therefore estimate the redox state o f QA a n d the e n e r g i z a t i o n of the t h y l a k o i d m e m b r a n e d u r i n g the i n d u c t i o n phase of photosynthesis. W h e n the conc e n t r a t i o n o f p h o s p h a t e in the m e d i u m is p r o p e r l y adjusted, intact isolated chloroplasts exhibit u p o n i l l u m i n a tion, a steady decline o f c h l o r o p h y l l fluorescence after the first m a x i m u m , c o n c o m i t a n t with a g r a d u a l increase in the rate of oxygen e v o l u t i o n (Fig. 1). The redox-stated e p e n d e n t chlorophyll fluorescence q u e n c h i n g (qp) a n d n o n - p h o t o c h e m i c a l chlorophyll fluorescence q u e n c h i n g (qN) increased in parallel, which is a characteristic of chloroplasts devoid o f a p r o p e r lag period (Cerovi6 1988). Once the m a x i m a l rate o f oxygen e v o l u t i o n was reached, qN c o n t i n u e d to increase while qp started to

S. Veljovi6-Jovanovi6 and Z.G. Cerovi6: Oscillations in isolated chloroplasts

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Fig. 3. The effect of the duration of the dark period preceding re-illumination on chlorophyll fluorescence in intact pea chloroplasts (conditions as in Fig. 1). When the maximum rate of oxygen evolution was reached (after 5 rain of illumination) chloroplasts were darkened for 10 s and then re-illuminated for 90 s. Chlorophyll fluorescence during the 90-s-light period is shown in the top trace. Five more experiments each with a different duration of the dark period (indicated in seconds n e x t to the traces) preceding re-illumination are presented in the other traces decrease as the rate of oxygen evolution started to decline. This behavior can be explained by the build-up of unfavorable inorganic versus organic phosphate in the surrounding medium (Lilley et al. 1977; Fliigge et al. 1980; Marques et al. 1987). Because of a fast depletion of inorganic phosphate and accumulation of organic phosphate in the surrounding medium, the m a x i m u m rate of photosynthesis is sustained only for a few minutes. The ratio between inorganic and organic phosphates constantly changes from being too high at the beginning of illumination, to being too low after prolonged illumination of intact isolated chloroplasts in vitro (Lilley et al. 1977). This is when intermittent illumination was applied with 20-s dark and 90-s light periods (Fig. 2). During the second period (first re-illumination m a r k e d 2 in Fig. 2) oscillations in chlorophyll fluorescence are hardly discernible, but became fully expressed during the fourth period. They vanish at the seventh period during which the oxygen-evolution rates has reached a low value. The experiment was reproducible but depended on

399

the "quality" of chloroplasts and the time they had spent on ice. In Fig. 3. only the second illumination period is presented, but following different durations of the preceding light period. As In Fig. 2, chloroplasts re-illuminated after they had reached the maximal rate of photosynthesis and darkened for 20 s, did not exhibit oscillations. Still, when the dark period was increased to 40 s, under otherwise identical conditions, chloroplasts started to show oscillations in chlorophyll fluorescence. This indicates that oscillations in photosynthesis can be induced either when the surrounding medium (Fig. 2) or the internal metabolite status (Fig. 3) has changed. At this point externally added metabolites, phosphoglycerate, dihydroxyacetone phosphate or inorganic phosphate at sub-millimolar concentrations abolished the oscillations (data not shown). As already noticed for algae (Bannister 1965), and protoplasts ( F u r b a n k and H o r t o n 1987), the oscillations presented here for isolated pea chloroplasts could be induced mainly when there was a restriction of photosynthesis under otherwise saturating conditions in respect to light and carbon dioxide. The influence of the phosphate status was also predominant, indicating an increased susceptibility to oscillate when the concentration of inorganic phosphate is low. Oscillations could be induced only at temperatures higher than 25 ~ C, which is the optimal temperature for photosynthesis measured for these pea chloroplasts (data not shown). This finding is in line with the notion that oscillations are present primarily when external factors are not limiting. The demonstration of the presence of oscillations of photosynthesis in isolated chloroplasts does not necessarily mean that they are generated by the same mechanism as those in vivo. The period o f oscillations is shorter than that usually detected in leaves (Ogawa 1982; Walker et al. 1983a, b; Stitt 1986; Peterson et al. 1988; Stitt et al. 1988; Stitt and Schreiber 1988) but is similar to that seen in algae (around 10 s) (Bannister 1965) or protoplasts in which cyclic p h o t o p h o s p h o r y l a t i o n has been inhibited ( F u r b a n k and H o r t o n 1987). Detailed biochemical and biophysical studies need to be done before any final conclusion can be drawn, but nevertheless, our results give additional credibility to the proposed existence of an "oscillatory center" in the chloroplasts themselves (Ogawa 1982; Walker et al. 1983a, b; F u r b a n k and H o r t o n 1987; Laisk and Walker 1989; Cerovi6 1988). The authors wish to thank Dr. Marijana Plesni6ar (Institute of Field and Vegetable Crops, University of Novi Sad, Novi Sad, Yugoslavia) who initiated this work, Professor David Walker (Department of Botany, University of Sheffield, UK) for stimulating discussions, and Ljubinko Jovanovi6 (INEP, Belgrade, Yugoslavia) for his help in the preparation of the manuscript. This work was supported in part by the Fund for Sciences of the Republic of Serbia. Z.G.C. was recipient of a bursary from the Commission of the European Communities at the time the manuscript was prepared.

References Bannister, T.T. (1965) Simple oscillations in photosynthetic oxygen evolution. Biochim. Biophys. Acta 109, 97-107

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