PhotosynthesisResearch 50: 181-191, 1996. (~) 1996KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Control of Photosystem II in spinach leaves by continuous light and by light pulses given in the dark N i k o l a i G. B u k h o v 1,2, Christian W i e s e I , S p i d o l a N e i m a n i s 1 & Ulrich H e b e r 1 IJulius-von-Sachs-lnstitute of Biological Sciences, University of Wiirzburg, D-97082 Wiirzburg, Germany; 2Timiriasev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia Received 10 May 1996;acceptedin revisedform5 November1996

Key words: chlorophyll fluorescence, energy dissipation, light scattering, photosynthesis, state transition

Abstract

The light-induced induction of components of non-photochemical quenching of chlorophyll fluorescence which are distinguished by different rates of dark relaxation (qNf, rapidly relaxing and qNs, slowly relaxing or not relaxing at all in the presence brief saturating light pulses which interrupt darkness at low frequencies) was studied in leaves of spinach. After dark adaptation of the leaves, a fast relaxing component developed in low light only after a lag phase. Quenching increased towards a maximum with increasing photon flux density. This 'fast' component of quenching was identified as energy-dependent quenching qE. It required formation of an appreciable transthylakoid ApH and was insignificant when darkened spinach leaves received 1 s pulses of light every 30 s even though zeaxanthin was formed from violaxanthin under these conditions. Another quenching component termed qNs developed in low light without a lag phase. It was not dependent on a transthylakoid pH gradient, decayed exponentially with a long half time of relaxation and was about 20% of total quenching irrespective of light intensity. When darkened leaves were flashed at frequencies higher than 0.004 Hz with I s light pulses, this quenching also appeared. Its extent was very considerable, and it did not require formation of zeaxanthin. Relaxation was accelerated by far-red light, and this acceleration was abolished by NaF. We suggest that qNs is the result of a so-called state transition, in which LHC II moves after its phosphorylation from fluorescent PS II to nonfluorescent PS I. This state transition was capable of decreasing in darkened leaves the potential maximum quantum efficiency of electron flow through Photosystem II by about 20%.

Abbreviations: PFD - photon flux density; PS - photosystem

Introduction

In photosynthesis, light is used to drive endergonic redox reactions. Water is oxidized and different acceptors such as CO2, nitrite, sulfate or oxalactetate are reduced. Excessively absorbed light energy is mainly dissipated as heat, but a very small fraction of it is emitted as fluorescence by the pigment system of PS II of the chloroplast electron transport chain. Photosynthetic energy conservation, fluorescence and heat dissipation are subject to regulation. When light is limiting, the quantum yield of carbon assimilation may reach

a maximum value of 0.125. Under excessive irradiation, it is very low. Owing to the competitive nature of photosynthetic electron flow, fluorescence and heat dissipation, fluorescence will decrease (be quenched) when electron flow increases, but also when heat dissipation increases. That part of fluorescence quenching which is caused by the dissipation of light energy as heat has often been termed qN (Krause and Weis 1991). By preventing excessive reduction of electron carriers and by decreasing the formation of reactive and potentially damaging radicals, qN is important for

182 the protection of the electron transport chain against photoinactivation (Horton et al. 1996). qN has several components (Horton and Hague 1988; Krause and Weis 1991). So-called energydependent fluorescence quenching qE depends on the magnitude of the transthylakoid proton gradient which is formed during linear or cyclic electron transport (Briantais et al. 1979; Sch6nknecht et al. 1995). Another qN component is caused by a so-called state 1/state 2 transition, in which the phosphorylation of fluorescent light harvesting complexes associated with PS II initiates migration of the complexes to non-fluorescent sites at P S I (Bonaventura and Myers 1969; Williams and Allen 1987; Horton and Black 1981). Finally, photoinactivation of PS II leads to a non-fluorescent state of PS II (qI) which is poorly characterized (Krause 1988, 1994). qI may have two components (Leitsch et al. 1994). For the analysis of fluorescence quenching, isolated thylakoid membranes, intact chloroplasts and leaves have been used. Several observations suggest loss of regulatory properties during the isolation of membranes or chloroplasts from leaves. For instance, expression of qE has been reported to be proportional to thylakoid acidification in vitro (Briantais et al. 1979; Krause et al. 1983), but qE rises steeply in vivo, by responding to a cooperative protonation reaction (SchOnknecht et al. 1995), after a threshold proton gradient has been built up. Electron transport is rapidly photoinactivated under high intensity illumination in isolated thylakoids and chloroplasts, but not in leaves (Krause 1988). In chloroplasts, elimination of qE by uncoupling has been used successfully to investigate the contribution of the state transition to total fluorescence quenching (Horton and Black 1981; Horton and Hague 1988). In leaves, this approach is not possible, and kinetic analysis of the relaxation of fluorescence quenching had to be used to distinguish quenching by qE, the state transition and qI (Horton and Hague 1988; Demmig and Winter 1988; Hodges et al. 1989; Quick and Stitt 1989; Waiters and Horton 1991; Lokstein et al. 1994). In the present communication, we use the traditional kinetic and also a novel approach to increase understanding of the role of the state transition in the control of PS II in leaves.

Materials and methods

Spinach (Sipinacia oleracea L.) was grown for 6 to 7 weeks in a greenhouse under short day conditions or was taken from a field. Prunus laurocerasus was from a garden location. Mature detached leaves well supplied with water through the petiole were used for the experiments. Before the measurements, the leaves were first exposed to low light and then dark-adapted for up to 12 h. Modulated chlorophyll fluorescence was measured as described by Schreiber et al. (1986) using the PAM system of Walz, Effeltrich, Germany. In most, but not all experiments, the modulated red measuring beam (modulation frequency 1.6 kHz) was present even if the leaves received no actinic light. It had a very low intensity (below 0.15/zmol m -2 s - l ) which itself had no measurable actinic effect. No difference was observed between this situation and when complete darkness was interrupted only by brief periods, in which the measuring beam recorded the effect on fluorescence of 1 s pulses of strong light. In some experiments a monochromator provided also a measuring beam of green light (505 nm) of an intensity which was even lower than that of the red measuring light. Red light of a half bandwidth ranging from 610 to 750 nm (combination of filters RG 610 from Schott, Mainz, Germany, and Calflex X from Balzers, Liechtenstein) or from 630 to 750 nm (filters RG 630 and Calflex X) was used for the actinic beam. A beam of far-red light (filters RG 9 from Schott and Calflex C) had a half bandwidth ranging from 710 to 740 nm. Light below 700 nm was negligible with this filter combination. Absorbed quanta of far-red light were 25 #mol m -2 s - l (measured in an Ulbricht sphere with the spectroradiometer 1800 of Licor, Lincoln, Nebraska, USA). Repetitive 1 s pulses of strong white light (PFD = 8500 #mol m -2 s - l ) were used to measure fluorescene quenching. When fluorescence and 505 nm absorption were simultaneously measured, red light pulses of 1 s duration (PFD = 3500/~mol m -2 s -1) were used instead of white light. 505 nm light was measured by a photomultiplier which was protected against actinic light and light pulses by two 9782 filters from Coming (Coming, NY) and a BG 18 filter from Schott (Mainz, Germany). For fluorescence parameters, the nomenclature of van Kooten and Snel (1990) was used. Nonphotochemical quenching qN was calculated as (Fro - Ftm)/(Fm - Fo), where (Fro - Fo) is the maximal pulse-initiated variable fluorescence yield of a predarkened leaf and F~m the pulseinitiated variable fluorescence yields observed during the subsequent illumination and darkening periods.

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Components of fluorescence quenching Figure 1 shows modulated chlorophyll fluoresence of a previously dark-adapted spinach leaf which received a PFD of 1050 #mol m -2 s -1 (equivalent to about one half of sunlight) for 5, 30 and 300 s. 1 s light pulses of very high intensity (8500 #mol m -2 s -1, about 5 times full sunlight) were given before, during and after actinic illumination as shown by transient increases (spikes) in maximum fluorescence yield Fm. They were intended to saturate photochemical energy conservation by fully reducing the primary quinone acceptor of PS II, QA, so that the difference between the height of the first spike and that of the following spikes could be used as an indicator of increased energy dissipation in the form of heat. Pulse-induced spikes were consistently higher before than during and after actinic illumination indicating extensive non-photochemical fluorescence quenching in the light. Recovery of large spikes in the dark was fast after short illumination periods and more slow after prolonged illumination. The increase in the height of the pulse-induced fluorescence spikes after darkening indicates qN relaxation. If relaxation were to obey first order kinetics, a plot of the logarithm of relaxation against time should yield straight lines. This expectation has been used by Horton and Hague (1988), Waiters and Horton (1991)

and others to analyze different components of fluorescence quenching. Figure 2 shows plots of lg ((FmF'm)/(Ftm-Fo) x 100) versus time of darkening after different periods of exposure of the leaves to a PFD of 810 #mol m -2 s -1 . (Fm - Fo) is the fluorescence spike induced by 1 s light pulse in a dark-adapted leaf and (Fm-F'm) the extent of nonphotochemical quenching persisting after actinic illumination. The plots of Figure 2 illustrate the complexity of nonphotochemical fluorescence quenching. A fast relaxing component observed after an illumination time of 30 s had a half time of relaxation of about 20 s, which was similar to relaxation of ApH-dependent quenching (Horton and Hague 1988). This component was particularly small after a shorter illumination period (10 s). Relaxation was slower when the illumination time was prolonged. An increase of the half time of fast relaxation was previously observed in Dunaliella cells (Lee et al. 1990). It was ascribed to retarded ApH dissipation. After a 180 s illumination period the half time of 'fast' relaxation was about 80 s. It was followed by a slower relaxation phase with a half time of several min. Full relaxation was not observed within the period of relaxation analysis. In fact, quenching increased during darkening, when the preceding illumination period had been very short (10 and 30 s). This effect was traced to quenching that developed in the dark as a consequence of the 1 s high intensity light pulses which were used to probe for quenching relaxation.

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Thus, two main phases of quenching can be distinguished on the basis of Figure 2. A fast relaxing phase qNf dominated quenching after light exposures longer than 10 s. It was attributed to energy-dependent quenching qE (Briantais et al. 1979; Krause et al. 1983; Quick and Stitt 1989) Waiters and Horton 1991). A much slower phase relaxing in the dark with a half time of several min is suggested to, be produced by quenching resulting from the phosphorylation of the light-harvesting complex LHC II (Bennet 1980; Black et al. 1984; Horton and Hague 1988). This quenching actually increased slowly in the dark when illumination periods had been shorter than. 60 s. It was traced back to the light pulses which were given in the dark to probe for quenching and remained stable in the dark as long as light pulses interrupted the dark period. Full relaxation required complete darkness. The quenching stabilizing about 10 min after actinic illumination close to log qN = 1.3 in Figure 2 is termed qNs. Figure 3 ,shows the development of qNf and qNs as a function of exposure to three different PFDs. The quenching analysis was performed according to Waiters and Horton (1991). At PFD = 49 #mol m -2 s - l , qNf first increased in a sigmoidal fashion with increasing illumination time. Subsequently it decreased. This should be expected, if development of quenching

depends an a large transthylakoid proton gradient. At the onset of illumination, existing photosynthetic substrates such as 3-phosphoglycerate are reduced (Heber et al. 1967) preventing the fast formation ofa transthylakoid proton gradient. As shown by light scattering measurements, this gradient becomes large in the presence of low PFDs only after readily available substrates have been consumed (Heber 1969). Once lightregulated photosynthetic enzymes such as fructose bisphosphatase have been activated (Buchanan 1980), 3phosphoglycerate becomes available again as a consequence of Calvin cycle activity, photosynthetic energy consumption increases and the transthylakoid proton gradient decreases. This explains the secondary relaxation of qNf in the presence of PFD = 49 # m o l m -2 s- i. At the higher PFDs of 260 and 810 #mol m -2 s- ] the secondary decrease in qNf cannot be seen because in these cases the energy input by light is larger than the energy consumption. The slowly relaxing phase qNs contributed about 20% to total qN irrespective of light intensity indicating that this phase did not fully saturate at low PFDs (see, however, Horton and Hague 1988). Importantly, it developed very rapidly (half time of about 10 s) and no lag phases were observed for qNs as for qNf at PFD = 49 and 260 #mol m -2 s -1 . Obviously, qNs did

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Figure 3. Developmentof slow and fast phases (qNs and qNf) of non-photochemical fluorescencequenching qN during illumination as calculated from (Fm,initia I - Fmt))./Fm,initial - F o ) during subsequent darkening(seeFigure 2). Illuminationof leaves with 49 (left), 260 (middle) and 810/~mol m-2 s- l (right). Each data point is the averageof 4 measurementsdone with differentdark-adaptedleaves. not depend on the formation of a large transthylakoid proton gradient. Waiters and Horton (1991) have proposed that the component of qN relaxing in the dark within several minutes has a complex nature and contains contributions from both state transition and qE. In order to characterize slowly relaxing quenching in the absence of a rapidly relaxing component, experiments were performed in which a leaf was exposed to 1 s pulses of intense red or white light given at low frequencies in the absence of continuous actinic light. Between pulses, only low measuring light recorded modulated fluorescence and, in some cases, 505 nm absorption changes. They had no measurable actinic effect. Practically identical observations were made, when light pulses were separated by periods of complete darkness.

Fluoresence quenching caused by short pulses of light Figure 4 shows simultaneous recordings of modulated chlorophyll fluorescence and 505 nm absorption for a spinach leaf which before the experiment had been

exposed to dim light for about 2 h and then to darkness for more than 10 h to deplete it of zeaxanthin (Demmig-Adams 1990). During a first 20 min period of light pulsing at the frequency of 0. 033 Hz (two I s pulses per min), maximum pulse-induced fluorescence declined by 26% in the absence of changes in 505 nm absorption. Importantly, the addition of 20% CO2 to the air stream passing over the leaf failed to initiate another slow phase of Fm decline. There was only a small drop both in Fm and Fo immediately after the CO2 had equilibrated with the leaf tissue. 505 nm absorption increased in response to high CO2. The kinetics of CO2-induced absorbance changes at 505 nm consisted of a fast and a slow phase. Very rapid responses to the light pulses are caused by light leaks. They can be neglected. In the absence of light pulses, slow 505 nm absorbance; changes did not occur (data not shown). It is known that acidification of the thylakoids (as it is expected to occur in the presence of high concentrations of CO2) triggers the de-epoxidation of violaxanthin to zeaxanthin (Hager 1969). Zeaxanthin absorbs more strongly at 505 nm than violaxanthin. This has

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been used repeatedly to measure zeaxanthin formation in the light (Bilger et al. 1989; Bilger and Bj6rkman 1990, 1994; Ruban et al. 1991; Veljovic-Jovanovic et al. 1993). Dithiothreitol (DTT), which is known to inhibit formation of zeaxanthin from violaxanthin (Yamamoto and Kamite 1972; Demmig-Adams et al. 1990; Bilger and Bj6rkman 1990, 1994), when fed to a leaf through the petiole, largely abolished the slow absorption increase seen in the presence of 20% CO2 and the slow decline observed when CO2 was subsequently removed• Only fast absorbance changes were similar in a DTT-fed leaf (data not shown) and in the experiment shown in Figure 4. We conclude that the slow increase in 505 nm absorption observed in the presence of 20% CO2 reflects the formation of zeaxanthin from violaxanthin, whereas the subsequent slow decrease in air indicates the slow reversal of this process. Since the existence of a close correspondence between zeaxanthin formation and fluorescence quenching under many different experimental conditions has suggested a direct and specific relationship between zeaxanthin and non-photochemical fluorescence quenching (Demmig-Adams 1990; Demmig-

Adams et al. 1992, 1996, Gilmore et al. 1994), the absence of such correspondence in the experiment of Figure 4 is remarkable. Horton et al. (1996) conclude that there is a common mechanism involved with and without zeaxanthin. In thylakoids lacking zeaxanthin high levels of quenching can be observed, which provides evidence that there is no obligatory mechanism linking zeaxanthin to quenching. Interestingly, pulse-induced maximum fluorescence declined again after CO2 was removed. The ratio of pulse-induced fluorescence to maximum fluorescence, which is a relative measure of the quantum efficiency of electron flow through PS II (Genty et al. 1989, 1990), had declined from an initial value of 0.8 to 0.65 in the minimum of fluorescence after the first acidification phase. When acidification by CO2 was repeated, qN actually relaxed somewhat in the presence of 20% CO2 after an initial small increase. Replacing high CO2 by air for a second and a third time in the experiment of Figure 4 caused an initial fast increase in pulse-induced fluoresence and then once again a slow decline towards a minimum. At the minima of fluorescence in air, qN was 0.5 after the first acidification

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phase, 0.58 after the second and 0.61 after the third phase. This shows considerable non-photochemical fluorescence quenching and, simultaneously, of control of PS II activity which was produced by short light pulses given to an essentially darkened leaf in air. The extent of both effects decreased as the frequency of light pulsing was decreased (see also Figure 6).

qNf in darkened leaves caused by short pulses of light Energy-dependent fluorescence quenching is known to depend on a low intrathylakoid pH and to relax when the intrathylakoid pH increases. The initial fast increase in pulse-induced fluorescence observed when high CO2 was replaced by air in the spinach experiment of FigUre 4 is reminiscent of fast qNf = qE relaxation. Figure 5 shows an experiment similar to that shown in Figure 4, but performed with a leaf of Prunus laurocerasus. Light pulsing at the frequency of 0.03 Hz in air decreased pulse-induced fluorescence and failed to affect 505 nm absorbance as it had done in spinach, but in contrast to the fluorescence observations made with spinach, 20% CO2 caused additional slow fluorescence quenching in Prunus. 505 nm absorbance increased even more in 20% CO2 trial it did in spinach. When. CO2 was replaced by air, pulse-induced fluorescence increased rapidly in Prunus, while 505 nm absorbance decreased slowly. The slow fluorescence decline in

CO2 and the fast increase in air is evidence that high CO2 had caused appreciable energy-dependent fluorescence quenching in Prunus. By comparison, the data of Figure 4 suggest that qNf = qE is only a minor component of the fluorescence quenching observed in the spinach experiment. The observation that slow fluorescence quenching did not only not increase in the presence of high CO2 in Figure 4 but actually relaxed somewhat at least during the second and third acidification phase must be considered as evidence that the quenching, which first increased in air and then started to relax in air, continued its slow relaxation in high CO2 while some qE was formed. Thus, one quenching component relaxed slowly in high CO2 while another one was slowly formed.

Effects of far-red light on pulse-induced fluorescence quenching in air When continuous far-red light replaced darkness in air in the Prunus experiment of Figure 5, maximum fluorescence decreased initially and then increased slowly. Far-red light is known to reverse state 1/state 2 transitions thereby increasing chlorophyll fluorescence from Photosystem II (Bonaventura and Myers 1969; Murata 1969). In the spinach experiment of Figure 4, farred light added after the third acidification phase in air increased pulse-induced maximum fluorescence rapidly at first and then more slowly. When the pulse frequency was doubled to 0.066 Hz, another large increase was observed. Turning the far-red beam off abolished the fluorescence increase. There was, the question whether the quenching of pulse-induced maximum fluorescence observed in air after the acidification phases in Figure 4 is related to the transient reduction of electron carriers which can be seen as increased steady state fluorescence (Allen et al. 1981). An experiment very similar in principle to that shown in Figure 4 was therefore performed, but far-red light was present throughout the experiment. In the presence of far-red, the transient reduction of electron carriers seen in the CO2/air transition in Figure 4 was absent. Nevertheless, quenching of pulse-induced maximum fluorescence was still observed in air and after high CO2 was replaced by air (data not shown) dearly indicating that this quenching does not require the high level of reduction of electron carriers which is apparent during the CO2/air transitions in Figure 4. Figure 6 explores the relationship between the frequency of light pulses and the extent of pulse-induced formation of qN in a darkened leaf. Very little pulse-

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Figure 7. Effects of intense short light pulses (1 s, 3500/zmol m - 2 s - l ) given every 30 s in air or 30% C02 (in air) on modulated chlorophyll fluorescence of a dark-adapted spinach leaf which had been fed lO mM dithiothreitol for 100 min through the petiole. Farred light (25/zmol m - 2 s - 1 absorbed quanta) was added as indicated by hollow arrows.

induced quenching was seen when the frequency of pulsing was below 0.004 Hz ('dark' times between the pulses longer than 4 min). Far-red background illumination largely abolished the pulse-induced qN at low (Figure 6), but not at high pulse frequencies (data not shown). In the experiment of Figure 7, a predarkened spinach leaf was fed 10 mM dithiothreitol through the

petiole for 100 min in the dark in CO2-free air (to prevent stomatai closure). Pulses of red light in air given to the darkened leaf decreased maximum fluorescence. This effect was reversed in far-red light. Importantly, acidification by 30% CO2 stimulated the reversal of fluorescence quenching by far-red light. The response of 505 nm absorbance (not shown) indicated that the dithiothretol had been effective to inhibit formation of zeaxanthin. When the far-red beam was turned off, maximum fluorescence decreased again, and this effect continued after high CO2 was replaced by air.

Fluoride inhibits reversal of quenching by far-red light In the experiment of Figure 8, a predarkened spinach leaf was fed 20 mM NaF through the petiole for 3 h in the dark in CO2-free air. Pulses of red light in air given to the darkened leaf decreased maximum fluorescence as they did in the dithiothreitol experiment of Figure 7. However, far-red light now failed to reverse this decrease as it did in Figure 7.30% CO2 decreased maximum fluorescence, and this decrease continued after the far-red beam was turned off. When high CO2 was replaced by air, maximum fluorescence stabilized at a decreased level. Surprisingly, 505 nm measure-

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ments indicated that not only dithiothreitol but also NaF inhibited zeaxanthin formation (data not shown). Since fluoride is known to inhibit the phosphatase which dephosphorylates light-harvesting complexes which are non-fluorescent in state 2, and dephosphorylation is required for transition to the fluorescent state 1, the data of Figure 8 are taken as evidence that most of the pulse-induced qN shown in Figures 4 to 8 is qNs and due to a state 1/state 2 transition. In cyanobacteria qN originates from a state-transition which changes the distribution of exitation energy between the photosystems (Campbell and t3quist 1996). The same was found for cyano-lichens by Sundberg et al. (1995).

Conclusions

In the leaves used for this work, epoxidation of zeaxanthin tO violaxalithin had occurred and key enzymes of the photosynthetic carbon cycle had been inactivated during darkening periods. Stomata were closed (if they were not forced to open during exposure of leaves to CO2-free air in the dark). Exposure to bright light was always short to prevent photoinhibition. Slow activation of carbon cycle activity in the light explains the secondary decline of rapidly relaxing qNf in the left part of Figure 3: Formation and maintenance of qNf require the presence of a considerable proton gradient, which declined, when it was used for the synthesis of ATP during carbon cycle activity, qNf was identified as energy-dependent fluorescence quenching which may

also be termed qE. It persisted only when light was permanently in excess. No evidence for qNf formation in air was obtained when spinach leaves received brief pulses of high intensity light which were interrupted by prolonged periods of darkness or of light of an intensity too low for an actinic effect (Figures 4-8). Some qNf formation was observed in this situation only when 20 or 30% CO2 caused cytoplasmic acidification and zeaxanthin was synthesized from violaxanthin even in the dark. The light pulses were fully sufficient for plastoquinone reduction. Reoxidation of plastoquinon in the dark is known to be slow (half time in the range of 2.5 to 5 min, Heber and French 1968). Plastoquinone reduction is thought to trigger a state 1/state 2 transition (Allen et al. 1981). However, we have also observed, that pulse-induced quenching of maximum fluorescence could only partially be inhibited by a background of far-red light if the pulse frequency was sufficiently high. Far-red also failed to suppress the pulse-induced quenching of maximum fluorescence quenching seen in spinach after a CO2/air transition even though it eliminated the transient reduction of electron carriers seen in Figure 4 as increased steady state fluorescence. Nevertheless, by its responses to far-red light and to NaF, most of the quenching caused by short light pulses in darkened leaves was identified as quenching resulting from the phosphorylation of light-harvesting complexes and the subsequent dissociation of the complexes from PS II. State 1/state 2 transitions are known to be reversed by far-red illumination (Bonaventura and Myers 1969; Murata 1969). Usually, statel/state 2 transitions are considered to be slow and to be saturated in low light (Chaw et al. 1981; Horton and Black 1981). Our method of analysis suggests that they can be fast in leaves (Figure 3), and that they rise steeply in low light and then less steeply as the light intensity is increased (Figure 3). Relaxation in the dark was very slow. It was accelerated by far-red light (Figures 4-7). This relaxation could be inhibited by the phosphatase inhibitor NaF (Figure 8), which is known to inhibit the far-red-induced reversal of state 2 to state 1 (Canaani et al. 1984; Quick and Stitt 1989). Depending on experimental conditions, the extent of fluorescence quenching by qNs was considerable (Figures 3 and 4). The relative quantum efficiency of electron flow through PS II was decreased by qNs from a maximum of 0.8 to 0.6 in the experiment of Figure 4. This figure may not yet reveal the full potential for control of PS II by qNs.

190 Zeaxanthin is believed to be involved in the formation of qE (Horton et al. 1994; 1995; Demmig-Adams et al. 1996). Both zeaxanthin synthesis and qE require a low intrathylakoid pH. In the experiment of Figure 4, acidification by CO2 was sufficient to trigger zeaxanthin synthesis but could not increase qN beyond the increase produced already in air. This shows that the quenching produced in the absence of high CO2 was not qNf = qE quenching, but qNs quenching. It also shows that the sensitivities to low pH of zeaxanthin formation and qE formation are not identical. However, they overlap. In some experiments with leaves of different origin or from other species (not shown), high CO2 was capable not only of inducing zeaxanthin synthesis but also of causing some additional fluorescence quenching which, as it requires acidification, is recognized to be qE quenching. It must finally be emphasized that the ATP-, dithiothreitol- and zeaxanthin- requiring quenching of chlorophyll fluorescence in darkened chloroplasts as reported by Gilmore and Yamamoto (1992) is mainly qE quenching brought about by thylakoid acidification during ATP hydrolysis, whereas the qNs quenching observed with darkened leaves in air in the present study does not appear to have a qE component.

Acknowledgments This investigation was performed within the framework of the research of the Sonderforschungsbereich 251 of the University of Wtirzburg.

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