Photosynthesis Research 11:119-130 (1987) © Martinus Nijhoff Publishers, Dordrecht - - Printed in the Netherlands

119

Regular paper

Sudden changes in the rate of photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts: the role of orthophosphate HITOSHI NAKAMOTO 1, MIRTA N. SIVAK* and DAVID A. WALKER Research Institute for Photosynthesis, University of Sheffield, Sheffield $10 2TN, UK (Received: 20 December 1985; in revised form: 7 March 1986)

Abstract. Simultaneous "ripples" (sudden changes in rate) in COs dependent 02 evolution and associated chlorophyU a fluorescence were followed in isolated, largely intact, spinach chloroplasts. These ripples could only be observed under conditions in which the supply of inorganic phosphate was limiting. This limitation was achieved either by 1) omission of phosphate in the assay medium, 2) use of inh~itors of the phosphate translocator, or 3) the addition of triose phosphate, a competitive inhibitor of Pi for the same translocator. The possible relation of these ripples to the dampening oscillations that can be observed in leaves, leaf pieces, isolated cells and protoplasts, is discussed.

Abbreviations Pi, orthophosphate; PPi: inorganic pyrophosphate; BSA: bovine serum albumin; EDTA: sodium ethylene-diaminetetraacetate; Hepes: 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid; DHAP: dihydroxyacetone phosphate; PGA: 3-phosphoglycerate. Introduction

The relation between chlorophyll a fluorescence kinetics and photosynthesis has been studied for many years, both in vivo and in vitro. Chlorophyll fluorescence yield is determined by more than one quenching mechanism (for reviews see [12, 14, 29]). One of them, qQ, is related to the redox status of the electron transport chain. The build-up of a transthylakoid proton gradient which occurs upon illumination is linked to another, qe [ 12]. The existence o f these two quenching mechanisms would be enough to result in the ambiguity (i.e. apparently similar kinetics displayed by photosynthetic systems in different states) and the complexity displayed by chlorophyll fluorescence in vivo, but several factors add even further to it. State 1-state II transitions, photoinhibition and stress also modify fluorescence [12]. The contribution of all of these to the fluorescence signals varies with the plant material and the conditions (light-intensity, CO2 and Oz concentration, temperature, etc., for reviews see ref. [12, 29]). 1Present address: Department of Botany, Washington State University, Pullman, 99164, USA *Address for offprints and correspondence: Mirta N. Sivak, Research Institute for Photosynthesis, The University, Sheffield S10 2TN, UK

120 The relation between chlorophyll fluorescence and photosynthesis has remained uncertain until recently, with some reports indicating a parallel and others an antiparallel relationship (see, for example, refs. [2, 6, 17, 19-22, 29, 40]). This uncertainty might have been due, in part, to methodological difficulties (c.f. refs [19] and [40]). However, the complexity of the fluorescence signal and its ambiguity have played, no doubt, a major role in this situation. The understanding of the relationship between photosynthetic carbon assimilation and fluorescence kinetics is still inadequate. Accordingly, the nomenclature of the fluorescence signal has been, mainly, descriptive [14]. As noted above, leaves can display what might look like similar fluorescence kinetics signals when the photosynthetic apparatus is, in fact, in very different states [27, 29, 34] and nomenclature has reflected this lack of understanding. New apparatus, methods and experimental approaches [12, 24, 29] have already started to change this situation in respect to chlorophyll fluorescence in vivo. Isolated chloroplasts usually display fairly simple chlorophyll fluorescence kinetics when compared with those of the parent tissue. In some circumstances, however, slow secondary kinetics can be observed and, as in leaves, it is tempting to assume that they reflect the same underlying phenomenon. Slow fluorescence kinetics displayed by isolated chloroplasts has been described by Carver [3, 5] and by Cerovic et at [6]. The similarity between the phenomena described by these authors, however, is only superficial. The transients described in [6] are associated with photosynthetic induction, i.e. the "shoulder" that interrupts the fail in chlorophyll a fluorescence after illumination is associated with the exponential increase in the rate of 02 evolution. During the first minutes of illumination the relationship between 02 and fluorescence is not antiparallel, and resembles in this respect (and in the association of the transients to photosynthetic induction) the transient that leads to the "M peak" which can, in some circumstances, be observed in vivo [27, 29, 34]. Similar interpretations, supported by measurements of other aspects of the photosynthetic process [27, 29] have been proposed for both phenomena. The transients described by Carver [3, 5] were different. In these experiments, a "ripple" in chlorophyll fluorescence was associated with another in 02 evolution. These transients were favoured by high pH and absence of (added) phosphate in the assay medium. This phenomenon attracted our attention because different sorts of relationships between fluorescence and photosynthetic carbon assimilation can also be observed in vivo as noted above. Moreover, dampening osillations in photosynthesis which can be observed in vivo (and in isolated cells and protoplasts) [10, 11, 19, 22, 26, 29, 40] are clearly affected by Pi supply [11, 13, 15, 29-31, 37-39]. We have undertaken further investigation of Carver's phenomena to see if these might be related to the events in leaves.

121 Materials and methods Spinach (Spinacia oleracea L. c.v. Virtuosa, Zwaan BV, Barendrecht, Holland) was grown for 6 weeks in water culture, in a glasshouse under sunlight and supplementary incandescent lamps as described previously [33]. Leaves were pre-illuminated for 30rain at 120Wm -2 and isolated chloroplasts were prepared following the method of Walker [33] followed by a further purification by isopicnic centrifugation (modified from [ 18] ) as follows. The plant material, about 60g of spinach leaves, was homogenized in 200ml semi-frozen grinding medium for 2 - 4 sec (Polytron blender) and the homogenate was squeezed trough two layers of muslin. The brei was then filtered through eight layers of muslin and one layer of cotton wool, and spun in a minifuge 2 (Heraeus Christ) from 0 to 1700g in 100sec. The pellet was resuspended in a small volume of resuspending medium and the chloroplasts were further purified by isopicnic centrifugation in Percoll. Continuous gradients were obtained by centrifugation of 34ml 50% v/v Percoll in resuspending medium, for 55 min, at --2°C, at 30000g, in a Sorvall SS34 angle rotor. Aliquots (3 ml) of the chloroplast suspension were then layered on the Percoll gradient and the tubes were centrifuged for 10 min, at 4300g, at --2 °C, in a Sorvall SS34 rotor. Intact chloroplasts, recovered near the bottom of the tube, were diluted using resuspending medium and collected as a pellet by centrifugation (950 g for 90 sec at 4 °C, Minifuge 2, Heraeus Christ). The pellet was then gently resuspended in resuspending medium to a final chlorophyll concentration of about 2 mg. ml-1. The grinding medium contained 330mM sorbitol, 5 mM MgC12, 10mM Na4P207 (PPi), 2 mM sodium D-isoascorbate and lg.1-1 bovine serum albumin (BSA). The resuspending medium contained 343 mM sorbitol, 1 mM MgC12, 1 mM MnC12, 2 mM sodium ethylene-diaminetetraacetate (EDTA), 2 g.1-1 BSA and 50 mM 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid (HEPES), pH 7.6. The chloroplast preparation used contained over 90% intact chloroplasts as measured by their inability to reduce ferrycianide in the light [16]. CO2 dependent 02 evolution by isolated chloroplasts was assayed, unless otherwise stated, at 20 °C in a reaction mixture containing 330 mM sorbitol, 1 mM MgC12, 1 mM MnC12, 2 mM EDTA, 50 mM HEPES, pH 8.0, 2000 units of catalase and 30/ag of chloroplast chlorophyll in a final volume of 1 ml. 02 evolution and chlorophylla fluorescence were measured simultaneously using oxygen electrode apparatus (Hansatech Ltd, Kings Lynn, England) described previously [6] with minor modifications. The light intensity was 200W.m -2 (red; half band width, 625 to 675nm, heat filter [Toshiba, Japan], K65 [Balzers, Liechstentein] and RG 610 [Schott, Mainz, Germany] ). Emission was defined by a suitable filter combination (740 nm interference filter [Balzers] ; filter 5030 [Coming, New York] and RG 715 [Schott] ) and measured using a photodiode (DT 500 Photop photodiode).

122 The oxygen concentration signal was electronically differentiated as described previously [7]. This was done in order to observe more clearly the changes in rate of oxygen evolution. Sudden changes can be detected directly in the 02 concentration traces only when rates are rather high (see, for example, [7, 8, 40] ). This is not possible when the rates are low, as when isolated chloroplasts are assayed in the conditions described below. The oxygen concentration traces are still shown in the figures, however, as they were used to determine the maximum rates (shown between brackets). Electronic differentiation of the 02 concentration signal increased the sensitivity and allowed the study of the "ripples" in 02 evolution and their association with chlorophyll fluorescence kinetics. They were not, however, used for quantitative purposes. Results We have confirmed Carver's [3, 5] findings. The single ripple observed by her in the absence of added Pi has been accentuated, presumably due to the high intactness (over 90%) of our chloroplasts preparations (see [6] for the effect of low intactness on the overall fluorescence kinetics). The inclusion of 5 mM PPi in the assay medium which we used accentuates the transients (Figure 1). In the assay conditions including PPi, and omitting Pi, at least two peaks following the initial fall in chlorophylla fluorescence could be observed. This improvement is specially relevant because it lends to comparisons with the "dampening oscillation pattern" observed in leaves [26, 29-31], leaf pieces [8, 35, 40] and protoplasts [22]. They are associated with corresponding antiparallel troughs in the rate of 02 evolution (Figure 1) and phase shifts were very small or absent. No transients could be observed in the presence of lO0/aM Pi (Figure 1). The sudden changes in rate shown in Figure 1 were initiated by illumination of the chloroplast suspension. Similar ripples can also be initiated by reillumination of the chloroplasts after a period of darkness, with 02 and fluorescence still keeping the same broadly antiparallel relationship (not illustrated). Sudden changes in the rate of 02 evolution and associated chlorophyll a fluorescence were dependent on the CO2 concentration (Figure 2). Although photosynthetic rate was not noticeably affected by varying the concentration of NaHCO3 from 10 to 1 mM, ripples were only observed at the higher concentrations used. Ripples can also be elicited in chloroplasts illuminated for a few minutes in the absence of exogenous CO2, by the addition of NaHCO3 (Figure 3). The simple fluorescence kinetics observed in the absence of added CO2 resembles those observed in vivo when leaves are illuminated in CO2-free air [28]. The addition of tO mM NaHCOa after several min illumination initiated several ripples, reminiscent of oscillations, in both O2 evolution rate and in the

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Figure 1. Sudden changes in CO2 (10mM NaHCO3) dependent O2 evolution and chlorophyll a fluorescence resembling in vivo behaviour (see for example [40] ) can be observed in isolated, largely intact isolated spinach chloroplasts ff Pi supply is strongly restricted. In the presence of 5 mM PPi and no added Pi, two ripples in the rate of 02 and associated, antiparallel transients in chlorophyll fluorescence could be clearly observed (middle). If 0.1 mM Pi was added (right) 02 and fluorescence displayed simple kinetics. Maximum rates of 02 evolution are indicated between brackets. The oxygen concentration signal was electronically differentiated in order to observe more clearly changes in the rate of oxygen evolution, as sudden changes cannot be detected directly in the 02 concentration trace (broken line) when rates are relatively low. Electronic differentiation of the 02 concentration signal increased the sensitivity and allowed the detection of ripples.

associated chlorophyll a fluorescence. During these sudden changes in rate, the relationship between 02 evolution rate and fluorescence is complex (c.f. Figure 1), but, after the first few seconds, again becomes antiparallel with very small, if any, phase shift. In the experiments shown in Figures 1 to 3, the absence of added Pi was a pre-requisite for the observation of sudden changes and the presence of PPi accentuated them. In the absence of PPi, the rate of 02 evolution was very

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Sudden changes in the rate of photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts: the role of orthophosphate.

Simultaneous "ripples" (sudden changes in rate) in CO2 dependent O2 evolution and associated chlorophyll a fluorescence were followed in isolated, lar...
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