Photosynthesis Research 34: 449-464, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Photosystem I-dependent cyclic electron transport is important in controlling Photosystem II activity in leaves under conditions of water stress Eva Katona*, Spidola Neimanis, Gerald Sch6nknecht & Ulrich Heber Julius-von-Sachs-Institut fiir Biowissenschaften der Universitiit, Mittlerer Dallenbergweg 64, D-8700 Wiirzburg, Germany Received 26 February 1992; accepted in revised form 9 September 1992

Key words: light scattering, photoinactivation, proton gradient, PT00 photooxidation, quenching of chlorophyll fluorescence, redox poising Abstract

Leaves of the C 3 plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO 2 and/or oxygen. In the absence of CO 2, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P515 signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO 2 inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background of red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO 2 levels were decreased to or below the CO 2 compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F o fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor QA in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.

Abbreviations: F, FM, F~, F~, Fo, F0-chlorophyll fluorescence levels; qbexc-quantum efficiency of excitation energy capture by open Photosystem II; dOpsi~ - quantum efficiency of electron flow through Photosystem II; P.~5 - field indicating rapid absorbance change peaking at 522 nm; PT00- primary donor of Photosystem I; Q A - primary quinone acceptor in Photosystem II; Q N - non-photochemical fluorescence quenching; Q q - photochemical quenching of chlorophyll fluorescence

* On leave from the Biophysics Department, University of Medicine and Pharmacy 'Carol Davila', 8 Eroii Sanitari Blvd., 76241 Bucharest, Romania.

450 Introduction

Electron transport in photosynthesis is coupled to the vectorial transfer of protons from the chloroplast stroma into the intrathylakoid space. The resulting transthylakoid proton gradient is used for, and dissipated by, the synthesis of the ATP required for carbon assimilation, photorespiratory carbohydrate breakdown and other processes. Most workers agree that three protons leave the thylakoids for each ATP synthesized, but even H+/ATP values up to 4.5 have been reported (Grfiber et al. 1987). Photosynthesis can proceed rapidly at low chloroplast ratios of ATP to ADP and of NADPH to NADP (Laisk et al. 1991). However, a large proton gradient has been shown to be important in controlling Photosystem II activity and in facilitating radiationless energy dissipation under conditions when excess light is absorbed by the chloroplast pigment system (Bj6rkman 1987, Baker and Horton 1987, Foyer et al. 1990, Genty et al. 1990, Krause and Weis 1991). Such control and the accompanying dissipation of excess excitation energy as heat are necessary to prevent full reduction of the chloroplast electron transport chain when stomata are closed under water stress. Such reduction would lead to the photodestruction of the photosynthetic apparatus, an effect, which can easily be demonstrated with isolated chloroplasts (Heber et al. 1989). In leaves, chloroplasts can reduce several electron acceptors. The proton/electron stoichiometry of linear electron transport is still not certainly known (Furbank et al. 1990). If Q-cycle activity at the level of the cytochrome b/f complex is obligatory (Rich 1988), H+/e is 3. Auxiliary reactions would not necessarily be required to provide the ATP needed for carbon assimilation or photorespiration of C3 plants, if the H+/ATP ratio is 3. If Q-cycle activity is facultative as several researchers assume (Moss and Bendall 1984, Ort 1986), such reactions are required. The assimilation of nitrate consumes ATP only when ammonia is incorporated into ketoglutarate, and a large proton gradient may be formed during nitrate reduction. A proton gradient also builds up during the slow oxygen reduction in the Mehler reaction and the accompanying reduction of peroxidatively produced

monodehydroascorbate (Asada and Takahashi 1987, Schreiber and Neubauer 1990). However, Heber et al. (1978) and Furbank and Horton (1987) have shown that, at high but not at low light intensities, carbon assimilation of intact chloroplasts or mesophyll protoplasts is inhibited by low concentrations of antimycin A, which inhibit cyclic, but do not inhibit linear electron transport. A proton gradient is formed, and ATP is synthesized during cyclic electron transport around Photosystem I (Tagawa et al. 1963, Arnon 1977, 1991, Arnon and Chain 1977, 1979, Hosler and Yocum 1985). A necessary requirement for cyclic electron flow is proper 'redox poising' which denotes a redox situation in which electrons are not drained from the electron transport chain whilst simultaneously full reduction of electron carriers is avoided. It is questionable whether such redox poising is possible when an oxidized chloroplast NADP system traps electrons during carbon assimilation of leaves (Laisk et al. 1991). On the other hand, under excessive light, and when leaves close their stomata under water stress and net photosynthesis is decreased together with nitrate reduction (Kaiser and F6rster 1989), increased reduction of the chloroplast NADP system may produce a redox situation which makes cyclic electron flow possible. In preceding publications, we have shown that under high intensity illumination, and in the complete absence of CO 2 from air, excessive reduction of the chloroplast electron transport chain cannot be avoided in brightly illuminated leaves of C3 plants (Wu et al. 1991, Heber et al. 1992). However, full photosynthetic control is shown by extensive oxidation of PT00 in the reaction center of Photosystem I, if photorespiratory CO 2 turnover is facilitated at a CO 2 concentration close to the CO 2 compensation point. In this communication, we wish to present evidence of thylakoid energization by cyclic electron transport in leaves of C3 plants, when linear electron flow is restricted by the availability of electron acceptors. Using a different approach, Harbinson and Foyer (1991) have also recently concluded that cyclic electron transport supports a large transthylakoid photon gradient when access of CO 2 to the photosynthetic apparatus is reduced. Although our conditions of demon-

451 stration are not identical with photorespiratory conditions of brightly illuminated water-stressed leaves, extrapolation of the observations made when linear electron transport is restricted by acceptor availability at low flux densities of absorbed light lead to the conclusion that thylakoid energization of leaves whose stomata are closed is supported by Photosystem I-dependent cyclic electron flow.

Materials and methods

Detached mature leaves of cabbage (Brassica oleracea var. oleracea) grown in a greenhouse were used for the experiments. Leaves were cut under water, and petioles were kept in water during the experiments. Part of the lamina of a leaf was enclosed in a sandwich-type cuvette which could be aerated. The composition of the gas atmosphere could be varied using mass flow controllers (Tylan Corporation, Eching, Germany). Water contents of the gas stream leaving the cuvette, and CO 2 contents of the gas streams entering and leaving the cuvette were determined using an infrared gas analyzer (BINOS, Leybold-Heraeus, Hanau, Germany). The relative humidity of the gas stream entering the cuvette was about 50%. The gas flow through the cuvette was 500 ml min -1 and the temperature between 20 and 25 °C. The upper surface of the enclosed part of leaf lamina could be illuminated through a window in the cuvette with a beam of either red or far-red light or with the two beams simultaneously. The red beam (filters RG 630 from Schott, Mainz, Germany, and K65 from Balzers, Liechtenstein) had a half-bandwidth ranging from 624 to 662nm. It excited both Photosystems I and II. The far-red beam (filters RG 610 and RG 715 or RG 724 from Schott and Calflex C or Calflex X from Balzers, as indicated in the legends to the figures) had a half-bandwidth ranging from about 714 to 766nm or from 725 to 752nm. It should be noted that filters of the same designation used in the previous publication (Heber et al. 1992) had slightly different half bandwidths. The far-red beam excited predominantly Photosystem I (Melis et al. 1987). A third beam (filters RG 630 from Schott and Calflex C from Balzers) with a

half-bandwidth ranging from 625 to 755 nm was given in the form of l s high intensity ( > 5 0 0 W m -2) pulses. An extremely weak (0.008 W m -2) monochromatic measuring beam recorded transmission of the leaf at various wavelengths in the green part of the spectrum. It was used to detect electrochromic absorption changes, which give information on the transthylakoid electric field and peak at 522nm (Junge and Witt 1968, Junge 1977) and changes in light scattering, which give information on the transthylakoid pH gradient and peak at about 535 nm (Heber 1969, Krause 1973, K6ster and Heber 1982, Bilger et al. 1988). Filter arrangements to protect the recording photomultiplier against actinic light were as reported previously (Heber et al. 1992). The electrochromic signal could also be measured at 535 nm by virtue of its fast kinetics which distinguished it from the slow light scattering changes. Modulated chlorophyll fluorescence was measured by the PAM fluorometer of Walz (Effeltrich, Germany) as described by Schreiber (Schreiber 1986, Schreiber et al. 1986). Fluorescence intensity levels were designated and the photochemical quenching parameter (Qq) was calculated from fluorescence traces according to van Kooten and Snel (1990); see also Schreiber et al. (1986). The non-photochemical quenching parameter (Q~) was defined and calculated according to Bilger et al. (1988) as relative decrease in the maximum fluorescence level. Quantum efficiencies for excitation capture by (riPe×c) and electron transport through PS lI (~ps H) were calculated according to Genty et al. (1989, 1990). P700 photooxidation was monitored measuring optical absorbance changes at about 830 nm by a PAM fluorometer which was equipped with a proper emitter-detector unit (Schreiber et al. 1988).

Results and discussion

Figure 1 shows changes in the apparent absorbance at 535 nm of a cabbage leaf which was kept in the dark (with only the dim green measuring beam on) or illuminated either with a beam of far-red light or a beam of red light, or with the two beams superimposed on one another. The oxygen concentration was varied from 1 to 21%,

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Fig. 1. Changes in apparent absorbance at 535 nm of a cabbage leaf produced by illumination with 4.2 W m --z (R1) or 8.4 W m -z (R2) red light, or by 100 W m -~ (FR) far-red light, or by a combination of R 1 and FR. The red light had a half-bandwidth ranging from 624 to 662 nm, while the far-red light had one ranging from 725 to 752 nm. R1 and FR were chosen so as to produce a comparable rate of CO 2 assimilation in an atmosphere containing 1% 02 and 500 ~11 -~ CO 2. (A) CO s was not added, and the oxygen content of the gas phase was varied between 21 and I%. (B) 35/zl 1-' CO 2 was present in the gas phase whose oxygen concentration was varied as in (A). For further explanation, see text.

with o r w i t h o u t 35/~11-1 C O 2 simultaneously present. T h e intensities of the different b e a m s were balanced in a separate experiment so as to p r o d u c e identical p h o t o s y n t h e t i c C O 2 u p t a k e in an a t m o s p h e r e containing 1% oxygen and 5 0 0 / z l 1-1 C O z. In this situation, photorespiration is suppressed. In the separate assimilation e x p e r i m e n t 100 W m -2 far-red light 2produced as m u c h C O 2 u p t a k e as 4 . 2 W m red light (0.047 nmol cm -2 s - l ) . This was taken to reflect the extent o f P h o t o s y s t e m II excitation by the far-red b e a m which served to excite p r e d o m i n antly P h o t o s y s t e m I. T h e p u r p o s e of the experim e n t o f Fig. 1 was to obtain information on light scattering, L i g h t - d e p e n d e n t changes in the scattering o f 535 n m light are k n o w n to indicate

c h a n g e s in the transthylakoid p r o t o n gradient ( K r a u s e 1973, K6ster and H e b e r 1982, Bilger et al. 1988, but see H e b e r et al. 1986 and Brugnoli a n d B j 6 r k m a n 1992 for precautions necessary in the interpretation o f 535 n m signals). I n Fig. 1A, experiments were p e r f o r m e d in the a b s e n c e o f C O 2, in Fig. 1B with 35/~11 - I C O 2 p r e s e n t in the gas stream. This C O 2 concentration is close to the C O 2 c o m p e n s a t i o n point which c o r r e s p o n d s to the intercellular C O 2 conc e n t r a t i o n in air, if water stress enforces complete stomatal closure ( C o m i c and Briantais 1991). A t the C O 2 c o m p e n s a t i o n point, p h o t o respiratory and respiratory C O 2 p r o d u c t i o n are as fast as the p h o t o s y n t h e t i c refixation of evolved C O z, but b o t h are much slower than light-satu-

453 was reversible on darkening. The difference spectrum of the faster part of the signal is broad covering both the range of the electrochromic shift P515 (Junge 1977) and of light scattering (Heber 1969). Figure 2 shows a similar difference spectrum (A2) measured in air with 500/~11-1 CO 2 in order to minimize the acceptor limitations introduced in the experiment of Fig. 1. Insert I A in Fig. 2 demonstrates that the kinetics of the signal seen at 522 nm follow close-

rated assimilatory electron flow in air. In the absence of C O 2 from the gas stream entering the cuvette, a considerable part of the respiratory and photorespiratory CO 2 will escape from the leaf so that light-dependent linear electron flow is decreased even when compared with electron flow at the CO 2 compensation point. Figure 1A shows that far-red light ( 1 0 0 W m -2) given alone in CO2-free air produced an increase in 535 nm absorption which

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454 ly the kinetics of the far-red induced oxidation and of the slow dark reduction of P700 and plastocyanin. The difference spectrum of the slow part of the 535 nm signal in Fig. 1A which was almost absent in 21% oxygen, but large in 1% oxygen, had a peak at 535 nm. It is very similar to the difference spectrum C 4 in Fig. 2, which is identified as a light scattering increase (Heber 1969). Red light (4.2 W m -2) given alone after a dark period in Fig. 1A produced no appreciable change in absorption. Doubling of the red light (to 8 . 4 W m - 2 ) , however, decreased transmission of 535 nm light. The difference spectrum of this signal was very similar to that of the slow light scattering increase caused by far-red light in the presence of reduced oxygen levels, but there was also a small component distinguishable by fast kinetics in the signal which extends the difference spectrum into the 520nm region of the P515 signal. When in Fig. 1A the red beam ( 4 . 2 W m 2) and the far-red beam (100 W m -2) were superimposed on one another, 535 nm transmission decreased far more than seen by doubling the red light. The difference spectrum was broad. It contained a slow light scattering component and a faster P515 component. The light scattering component could be almost eliminated by adding some CO 2 to 21% oxygen (Fig. 1B). An almost pure far-red-dependent P515 signal remained as shown in a different experiment in the difference spectrum of Fig. 2B. The increase in the signals seen in Fig. 1 when red and far-red beams were superimposed cannot be due to Photosystem II excitation as shown by a comparison with the signals brought about by the doubling of the red beam. It must be attributed to Photosystem I excitation by the far-red light. Far-red light can increase the transthylakoid proton gradient by supporting coupled cyclic electron transport. We conclude that the experiment shows cyclic electron transport in a C3 leaf. However, even the low concentration of 35 kdl 1 CO 2 suppressed light scattering in the presence of 21% oxygen (Fig. 1B), probably in part by disrupting the cyclic pathway, as during the interplay of assimilatory carbon reduction and photorespiratory carbon oxidation NADPH

is consumed. NADP drains electrons from the cyclic electron transport chain. Light scattering may also be decreased by decreasing the transthylakoid proton gradient owing to increased ATP consumption. It should be noticed that the light scattering signal can be almost completely quenched by decreasing the transthylakoid proton gradient to levels which are still able to support appreciable carbon assimilation (Heber 1969, Dietz et al. 1984). When photorespiratory carbohydrate oxidation was decreased by decreasing the oxygen concentration, light scattering increased both in the absence of CO 2 (Fig. 1A) and in its presence (Fig. 1B) indicating not only decreased ATP consumption but also increased cyclic electron transport as shown by a comparison of the light doubling experiments (8.4 W m -2 red light versus 4.2 W m-2 red plus 100 W m -2 far-red light). In the presence of only 1% oxygen and in the absence of CO: (Fig. 1A) excitation of Photosystem II by 8 . 4 W m - : red light (but not by 4.2 W m -z) was sufficient to reduce the electron transport chain to such an extent that cyclic electron flow (which is possible in principle also under illumination with red light, but which requires some oxidized plastoquinone) was impeded. This is shown by a secondary suppression of light scattering (i.e. the transthylakoid proton gradient) after the initial light-dependent increase. It should be noted that additional Photosystem I excitation by the far-red beam not only prevented such inhibition in the same experiment but also stimulated light scattering more than at the higher oxygen concentrations. Obviously, Photosystem I is capable of controlling Photosystem II so that excessive reduction of the electron transport system, which leads to an inhibition of thylakoid energization, is prevented. The experiment in Fig. 1 is a demonstration of 'poising' in leaves. In experiments with thylakoids, Arnon has demonstrated a requirement of cyclic electron transport for a balanced redox situation of the electron transport chain which was described as poising (Arnon et al. 1958). Cyclic electron transport requires oxidized electron carriers between the two photosystems and reduced carriers on the reducing side of Photosystem I. Neither full reduction of the chloro-

455 plast electron transport chain (Ziem-Hanck and Heber 1980) nor excessive oxidation of the chloroplast N A D P system (Laisk et al. 1991) will permit cyclic electron flow to occur. Figure 3 shows light scattering changes of a cabbage leaf in an atmosphere containing 15% oxygen and either no CO 2 (A) or 500 ppm CO 2 (B). Light used for continuous illumination was either red to excite both Photosystems II and I or far-red to excite largely Photosystem I, or a combination of both. From a third light source, high intensity red flashes lasting 1 s were given every 40 s both in the dark and during continuous illumination with red or far-red or red plus

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Fig. 3. Changes in apparent absorbance at 535 nm of a cabbage leaf produced by 100 W m 2 far-red light (FR) and three different intensities of red light (R: 2.5, 10 and 30 W m 2) or combinations of red and far-red light. 1 s flashes of a broad band of red light (600 W m 2 half-bandwidth from 625 to 755 nm) were given every 40 s. Half-bandwidths of red and far-red lights were as in Fig. 1, Arrows indicate onset and termination of illumination. (A) CO 2 was not added to the gas phase which contained 15% oxygen. (B) 500 tzl 1-1 CO 2 was present in the gas phase which contained 15% oxygen. For explanation, see text.

456 changes produced by the flashes relaxed in the presence of far-red light with biphasic kinetics (not clearly seen because of low time resolution). When continuous red light replaced the far-red, apparent absorption at 535 nm remained low at the lowest intensity (2.5 W m-2), but increased progressively in Fig. 3A, but not in Fig. 3B, as the intensity of the red light was increased to 10 and 30 W m 2. Since this increase, which was seen only in the absence, but not in the presence of CO2, had a difference spectrum which peaked at 535 nm, it was mainly caused by increased light scattering (Heber 1969) and indicated formation of an appreciable transthylakoid proton gradient. The combination of red and far-red light had in the absence of CO2, but not in its presence, large effects on the light-dependent changes in apparent absorbance particularly at the lower intensities of red light (Fig. 3A). As in Fig. 1, the changes in apparent absorption caused by red plus far-red light were larger than expected on the basis of additive effects. Both P5~5 and light scattering increased. On darkening, the fast relaxation of P5~5 permits to distinguish the electrochromic change from the more slowly relaxing light scattering change. It can be seen that light scattering changes were appreciable in the absence of CO 2 and largely absent in its presence (except for a small and transient light scattering signal seen when red light of 30 W m 2 was turned on). In the presence of CO2, photosynthetic energy consumption had suppressed the light scattering changes. Short red flashes produced very different effects in the absence and in the presence of CO 2. Positive and rapidly reversible spikes were caused by a transient increase in P515 (difference spectrum C 1 in Fig. 2). Large negative spikes seen only in the absence of CO 2 (Fig. 3A) had a broad difference spectrum which distinguished this signal from P5~5 and light scattering changes (Fig. 2 C2). Its response to changes in the composition of the gas phase suggest that it is related to a transient photoaccumulation of reduced ferredoxin. The negative spikes were followed by a slow light scattering increase (identified by the difference spectrum shown in Fig. 2 C4) which was maximal 10 to 20 s after a flash had been given. Transiently increased thylakoid energiza-

tion indicated by these scattering increases was particularly pronounced in the presence of a low intensity red background light. It was less conspicuous, when the background scattering approached light saturation. The flash-induced secondary increase in thylakoid energization observed in the absence of CO 2 was not only completely missing in the presence of CO 2 but actually replaced by a small flash-induced decrease in apparent absorbance suggesting flash-induced additional photosynthetic energy consumption. Upon darkening, the system slowly reverted to the state observed before illumination. Figure 4 shows simultaneous recordings of modulated chlorophyll fluorescence and apparent absorbance changes at 535 nm of a cabbage leaf. In Fig. 4A, the oxygen concentration was 10% and CO z was absent. The leaf was flashed every 40 s with high intensity red light (1 s) either in darkness or in the presence of continuous far-red or of red light. In some cases extra flashes were given outside this routine to probe for the state of leaf energization. As in the experiment of Fig. 1, the intensities of the red and far-red beams had been balanced in a separate experiment so as to cause comparable carbon assimilation in an atmosphere containing 500 t~ll -~ CO 2 and 1% oxygen. Flashes given in darkness caused a transient increase in the P5~5 signal (upper traces in Figs. 4A and B) and increased fluorescence transiently from the minimum level F 0 indicating full oxidation of the primary quinone acceptor QA of Photosystem II to a maximum level F M which indicates its full reduction. The initial ratio (F MF0)/FM = ~cxc was close to 0.67 in Fig. 4A indicating some loss of the quantum efficiency of excitation energy capture in Photosystem II owing to the previous history of the leaf (Genty et al. 1989). The maximum relative quantum efficiency is close to 0.8 (Bj6rkman and Demmig 1987). When far-red background light (134Wm 2) was given, apparent 535 nm absorption increased initially fast owing to the formation of a transmembrane field and then slowly owing to increased light scattering. Although the light scattering change indicated increased thylakoid energization, no energy-dependent fluorescence

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Fig. 4. Simultaneous recording of 535 nm absorbance changes (upper trace) and of modulated chlorophyll fluorescence (lower trace) of a cabbage leaf which was illuminated with far-red (134 W m -2, FR) or red (9.6 W m 2, R) background light. It also received brief (1 s) pulses of high intensity red light (600 W m-2). Half-bandwidths of red light and of flashes were as in Fig. 2. The far-red light had a half-bandwidth ranging from 714 to 766 nm. The intensities of the red and far-red beams were balanced so as to produce comparable CO 2 assimilation in 1% oxygen containing 500/xl 1-~ CO 2. CO2 uptake in this situation was 0.1 nmol m 2 s ~. Arrows indicate onset and termination of illumination. (A) The gas atmosphere contained 10% oxygen. CO 2 was absent. The leaf was flashed every 40 s. Additional flashes were given as indicated, o.a. = optical artifact. (B) Before the experiment, the leaf was illuminated for 1 h in a nitrogen atmosphere to produce some photoinhibition. The gas atmosphere was CO2-free air, and the leaf was flashed every min. For further explanation, see text.

q u e n c h i n g was i n d i c a t e d in Fig. 4 A by a decrease of m a x i m u m fluorescence levels ( F ~ ) u n d e r farr e d i l l u m i n a t i o n . This is surprising, b e c a u s e by i n c r e a s i n g n o n - r a d i a t i v e e n e r g y dissipation, such e n e r g i z a t i o n s h o u l d have d e c r e a s e d F ~ . H o w e v e r , a state t r a n s i t i o n i n d u c e d by far-red light m a y h a v e i n c r e a s e d F ~ ( H o r t o n et al. 1990) o b s c u r i n g the d e c r e a s e e x p e c t e d from t h y l a k o i d energization. B r i e f light pulses given o n top of the far-red b a c k g r o u n d light had c o n s i d e r a b l e effects. W h i l e

light s c a t t e r i n g i n c r e a s e d slowly after the flashes, c h l o r o p h y l l fluorescence d e c r e a s e d to a n d e v e n s o m e w h a t b e l o w the F 0 level in Fig. 4A. W h e n a s e c o n d flash was given a few s e c o n d s after the first flash while light scattering h a d r e a c h e d its m a x i m u m , the m a x i m u m fluorescence level F ~ c o r r e s p o n d i n g to full QA r e d u c t i o n was m u c h d e c r e a s e d s h o w i n g that c o n s i d e r a b l e thylakoid e n e r g i z a t i o n h a d d e v e l o p e d after the first flash. B r i e f d a r k e n i n g after a flash d e c r e a s e d fluoresc e n c e slightly b e l o w its initial m i n i m u m level

458 indicative of full oxidation of QA at the beginning of the experiment, demonstrating quenching of the 'dark'-level fluorescence. When the far-red background light was turned off, 535 nm absorbance decreased in two kinetic phases. The first phase was fast and indicated relaxation of an electrochromic component (P515) and the second was slow. It showed relaxation of the light scattering change. Surprisingly, F M decreased somewhat in the dark, probably in a reversal of the state transition. The transient fluorescence decline to a very low level seen after a flash in the presence of far-red light was absent in the dark, and flashing a second time shortly after another flash did not produce a decrease in the maximum fluorescence level as observed under far-red background illumination. Red background light corresponding to the far-red beam in its ability to excite Photosystem II produced only a very small P515 signal (Fig. 4A). Light scattering increased slowly while F~ levels decreased indicating increased non-radiative energy dissipation (which was not seen under a far-red background), but flashing did not induce much additional light scattering. QA was somewhat reduced under red illumination as seen by the steady-state fluorescence level F which was clearly above F 0. After QA was fully reduced in a flash, it was only slowly reoxidized as seen by the slow return of fluorescence to its steady-state level. A second flash placed shortly after a first flash failed to indicate increased energization by a decrease in the maximum fluorescence level or by light scattering. QA occurred slightly more reduced. The data recorded in Fig. 4A show clearly that under the conditions of the experiment flashinduced additional thylakoid energization can only be produced in the presence of far-red light which is known to excite Photosystem I. It is neither observed as a post-illumination event in the dark nor in the presence of red background light whose intensity has been matched to that of the far-red light so as to produce comparable Photosystem II excitation. The experiment shown in Fig. 4B is similar to that of Fig. 4A with the exceptions that the cabbage leaf had been illuminated before the experiment for 1 h under anaerobic conditions in order to cause partial photoinhibition of Photo-

system II and that the gas atmosphere was C O 2f r e e air. Apparently, this treatment not only produced some increase in the level of F 0 (not shown), but interfered also with the ability of reduced QA to reoxidize after a brief flash. This is indicated by the slow relaxation of fluorescence from the maximum level F M to its 'dark' level F 0. In contrast to the experiment of Fig. 4A where far-red background illumination failed to quench flash-induced F~ fluorescence although it increased thylakoid energization as indicated by increased light scattering, far-red light decreased F~ in the experiment of Fig. 4B by increasing non-radiative energy dissipation. After the flash, fluorescence decreased (much more clearly than in the experiment of Fig. 4A) below the F 0 level while light scattering increased transiently as it had done in the experiment of Fig. 4A. The observations made after the optical responses induced by far-red light had relaxed in the dark were similar to those made in the experiment of Fig. 4A. To investigate in more detail the state of leaf energization after a 1 s high intensity red flash, additional 1 s flashes were given different times after the first flash had induced a slow transient scattering response in a leaf which was illuminated with far-red background light (Fig. 5). Apparent absorbance at 535 nm and modulated chlorophyll fluorescence were recorded simultaneously. The gas phase contained 5% O 2 and no CO 2. The relative quantum efficiency of excitation capture ~exc of the leaf had decreased from about 0.8 to 0.55 as a consequence of previous anaerobic pretreatment in the light which had also caused some increase in the F 0 level of fluorescence. This increased level is given as a straight line in Fig. 5A. QA remained fairly oxidized under the far-red background light as shown by a comparison of F, F 0 and F~ levels. By fully reducing QA, the first flash produced a rise in fluorescence to the maximum level F~. ( 1 - Qq) = (F-F0)/(F~-F0) was 0.12. Disregarding some nonlinearity between (1 - Qq) and QA reduction, this shows that about 12% of Photosystem II reaction centers were closed by the far-red background light. It should be noted that no F 0 quenching was observed in the presence of background FR alone. Non-photochemical quenching in the presence of background

459

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Fig. 5. Flash-induced changes in 535 nm light scattering of a cabbage leaf (upper traces), of chlorophyll fluorescence lower left trace) and of fluorescence parameters (lower right: Flash-induced suppression of 'dark' fluorescence F, = 1 to the level F[~, flash-induced non-photochemical fluorescence quenching Q; = (F~-F~)/Fd; flash-induced changes in the redox state of QA as indicated by changes in the fluorescence parameter ( 1 - Q 0 ' (Fully reduced QA corresponds to (1 Qq) = t.) The flash (1 s, 600Wm 2) had a half-bandwidth ranging from 625 to 755nm. It was given on far-red background light (134Wm 2, half-bandwidth: 714-766 nm). Subsequent flashes were given after various times to probe for fluorescence changes induced by the first flash. An evaluation of these changes is compared with light scattering in (B) and an example of the procedure is shown in (A). The gas atmosphere contained 5% 0 2. CO 2 was absent. Before the experiment, the leaf was subject to photoinhibitory treatment as in the experiment of Fig 4B.

f a r - r e d was QN = ( F M - F ~ ) / F M = 0 - 1 6 . The q u a n t u m efficiency of e l e c t r o n flow t h r o u g h P h o t o s y s t e m II was q%s 11 = ( F ~ - F ) / F ~ = 0.50. A f t e r t h e flash, t h e a p p a r e n t a b s o r p t i o n at 535nm increased and fluorescence decreased b e l o w t h e F 0 level as it did in the e x p e r i m e n t s d e s c r i b e d in Fig. 4. T h e ' d a r k ' - l e v e l of the chlor o p h y l l f l u o r e s c e n c e a f t e r t h e flash, F~, was det e r m i n e d b y switching off t h e b a c k g r o u n d f a r - r e d light for s e v e r a l s e c o n d s at d i f f e r e n t t i m e s a f t e r t h e first flash (not shown). A s e c o n d flash ind u c e d a d d i t i o n a l light s c a t t e r i n g a n d c a u s e d fluor e s c e n c e to rise to F ~ (Fig. 5A). A r e l a t i v e m e a s u r e of QA r e d u c t i o n is then 1 - Qq = ( F -

F ~ ; ) / ( F ~ - F ( ; ) a n d a m e a s u r e of t h e f l a s h - i n d u c e d i n c r e m e n t in n o n - p h o t o c h e m i c a l f l u o r e s c e n c e q u e n c h i n g in O ; = ( F ~ - F d ) / F ~ . F i g u r e 5B s h o w s the d e p e n d e n c e of t h e s e f l u o r e s c e n c e s p a r a m e t e r s on the t i m e i n t e r v a l r b e t w e e n two flashes. It can be s e e n that an i n c r e a s e in light s c a t t e r i n g is a c c o m p a n i e d by an i n c r e a s e in nonphotochemical fluorescence quenching. Nonphotochemical fluorescence quenching declined while light s c a t t e r i n g d e c l i n e d . C h a n g e s in F 0 w e r e , as e x p e c t e d , a n t i p a r a l l e l to c h a n g e s in n o n - p h o t o c h e m i c a l fluorescence q u e n c h i n g and to light s c a t t e r i n g (see also Fig. 4 A a n d B). It is particularly remarkable that non-radiative ener-

460 gy dissipation in Photosystem II increased while reduction of QA decreased. Since the increased energization of the thylakoids indicated by increased light scattering is a Photosystem I effect (Figs. 1, 3 and 4), the data show clearly that Photosystem I can control Photosystem II not by preventing reoxidation of photoreduced QA by a large proton gradient but rather by controlling reduction of QA. In Fig. 6, 535 nm absorbance changes of a cabbage leaf and photooxidation of PT00 as indicated by absorbance changes in the 830 nm region were simultaneously recorded. As should be expected, the far-red background light (FR1) not only produced a Pst5 signal (upper trace, seen as fast increase in absorbance at 535 nm on illumination and as a fast decrease on darkening) and slower light scattering changes, but also considerable photooxidation of PT00 (lower trace). By exciting Photosystem II and transporting electrons from water into the intersystem chain between the two photosystems, the flashes resulted in a partial reduction of PT00÷. As PT00 was slowly reoxidized, light scattering increased. It decreased again when PT00 oxidation reached a steady state. It is important to note that this

FR1

steady state did not represent full oxidation. W h e n after darkening and illumination with short wavelength red light and a second darkening period a far-red b e a m (FR2) was turned on which not only had a lower intensity than the far-red b e a m used initially ( R G 715), but was also shifted further into the far-red, photooxidation of PT00 was increased by 10% c o m p a r e d with the oxidation observed initially. As with F R I , flashes reduced photooxidized PT00- However, reoxidation after the flashes was faster than in the presence of FR 1 background, although the flashes had produced comparable water oxidation by PS II, and not only Photosystem II but also Photosystem I excitation was reduced by a shift towards the far-red. Apparently, poising was altered and electrons escaped via a linear electron transport pathway instead being forced into the cyclic pathway. This is also shown by decreased light scattering and the absence of extra scattering after the flashes. In the presence of short wavelength red light, with PS II excitation comparable to that caused by FR1, the flashes failed to produce appreciable changes in the redox state of P700" m small flash-induced oxidation was transient. As in the experiment of

dark

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[ e~ J

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