using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photo- chemical quenching of chlorophyll fluorescence. (qo) and ...
Planta (1988)| 76:433-M40
P l a n t a 9 Springer-Verlag 1988
Regulation of electron transport in maize mesophyll chloroplasts: The relationship between chlorophyll a fluorescence quenching and 02 evolution Robert T. Furbank* Research Institute for Photosynthesis, University of Sheffield, Sheffield SI0 2TN, U.K.
Abstract. The relationship between the redox state of the primary electron acceptor of photosystem II (QA) and the rate of 02 evolution in isolated mesophyll chloroplasts from Zea mays L, is examined using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photochemical quenching of chlorophyll fluorescence (qo) and the rate of 02 evolution is evident under most conditions with either glycerate 3-phosphate or oxaloacetate as sabstrates. There appears to be no effect of the transthylakoid pH gradient on the rate of electron transfer from photosystem II into QA in these chloroplasts. However, the proportion of electron transport occurring through cyclicpseudocyclic pathways relative to the non-cyclic pathway appears to be regulated by metabolic demand for ATP. The majority of non-photochemical quenching in these chloroplasts at moderate irradiances appeared to be " energy "-dependent quenching. Key words: Chlorophyll fluorescence- Chloroplast (electron transport) - Electron transport - Oxygen evolution - Photosynthesis (electron transport) Photosystem II - Zea (electron transport)
Introduction
Measurement of room-temperature chlorophyll a fluorescence is now an established non-intrusive probe of photosynthetic processes in vivo and in * Present address: CSIRO, Division of Plant Industry, GPO
Box 1600, Canberra, A.C,T. 260I, Australia Abbreviations and symbols," PS[I = photosystem II ; F m = maxi-
mum fluorescence obtained on application of a saturating light pulse; Fo = basal fluorescence recorded in the absence of actinic light (i.e, all PSl [ traps are " o p e n "); Fv = Fm -- Fo; qQ = photochemical quenching; qr~e= non-photochemical quenching; qE = "energy"-dependent quenching of chlorophyll fluorescence
vitro (Bradbury and Baker 1981; Schreiber 1983; Sivak and Walker 1985), The technique of "light doubling" (Quick and Horton 1984; Schreiber et al. 1986), i.e. flashing with high-intensity light in addition to actinic light to fully reduce QA, the primary acceptor of photosystem II (PSII), permits the deconvolution of fluorescence quenching into two components: (i) photochemical quenching (qo), arising from the oxidation of QA and (ii) nonphotochemical quenching (q~e), the majority of which, in unstressed tissue, is generally accepted to be high-energy-state quenching (qr) arising from a high transthylakoid ApH (see Sivak and Walker 1985; Schreiber et al. 1986). Although measurement of these components of fluorescence quenching is now relatively simple, there are still some fundamental questions regarding the relationship between fluorescence quenching and electron transport which remain unanswered. Lavorel and Etienne (1977) suggested that the rate of electron flow may be assumed to equal the rate of charge separation in the PSI1 reaction centre which, in turn, should be linearly related to qQ. If this were the case, one would predict a simple linear relationship between photosynthetic assimilation rate and qo" This hypothesis is difficult to test in vivo because of the operation of energy-consuming reactions dependent upon electron transport but not necessarily resulting in net 02 evolution or CO2 fixation (e.g. Mehler reaction and photorespiration), These reactions have been held responsible for the observation that, in most intact-leaf experiments reported in the literature to date (Schreiber and Bilger 1987; Weis and Berry 1987; Weis et al. 1987), 02 evolution and COz fixation are not linearly related to q•. Recently, a new model has been proposed which could provide an alternative explanation for such deviations (Weis etal. 1987; Krause and Laasch 1987). This model does not
434
dispute that the rate of charge separation and the redox state of QA are closely related but proposes that the "intrinsic" efficiency of PSII reaction centres is indeed flexible. It is proposed that transthylakoid ApH may play a part in this regulation and that high ApH induces a low-fluorescence, low-quantum-yield reaction centre. This is evidenced by a decrease in the effective use of quanta by PSII reaction centres with increasing light, a process which is highly correlated with qNP (Weis et al. 1987). This is also supported by the observation that QA and PSI are relatively oxidised under all experimental conditions, even at high light intensity, indicative of secondary regulation of PSII rather than an accumulation of electrons at QA caused by insufficient electron acceptor (Weis et al. 1987). Alternatively, cycling of electrons around PSII as suggested by Schreiber and Reinits (1987) could also provide a mechanism for maintaining QA in the oxidised state at high light intensities. Schreiber and Reinits observe an ATP-dependent photochemical quenching of chlorophyll fluorescence which responds to the activity of the thylakold ATPase. They propose that this oxidation of QA is the result of cycling of electrons around PSII, possibly via low-potential cytochrome b559. Irrespective of the mechanism involved, these observations indicate that a process exists whereby the rate of electron transport could be controlled at PSII in a manner which is responsive to thylakoid ApH and hence, ATP demand. The possibility of such regulation is extremely important as it could provide both a mechanism to prevent over-reduction of the electron-transport chain under photoinhibitory conditions and the basis for a new type of "photosynthetic control" of electron transport under conditions where the ApH is excessive. The origin of high-energy-state quenching of chlorophyll fluorescence is currently a matter of debate. There is substantial evidence supporting the theory that qE is closely related to, if not a consequence of, formation of the transthylakoid pH gradient (Krause et al. 1982) but the biophysical basis for qE remains undiscovered. Recent evidence (Oxborough and Horton 1987) indicates that ApH and qE formation are not inseparable events in photosynthesis and that ApH may be formed in the absence of qE. Poor quantitative correlations between 9-aminoacridine fluorescence quenching and qNa were also recently found with intact spinach chloroplasts during CO2-dependent O2 evolution (Furbank et al. 1987). Also, as non-photochemical quenching is composed of a number of components other than qE, it is important to determine if qE is, in fact, the major component of non-
R.T. Furbank: Regulation of electron transport in maize
photochemical quenching in the experimental system used. The maize mesophyll system provides an opportunity to examine thylakoid ApH and the components of qNP in an intact, coupled chloroplast with endogenous electron acceptors. In this study, isolated maize mesophyll chloroplasts were used as an experimental system in which to examine the relationship between qo and the rate of O2 evolution, qNP and thylakoid ApH (measured by 9-aminoacridine fluorescence quenching). This system is particularly useful as both qNP and qo can be manipulated independently by the addition of substrates requiring differing ratios of NADPH/ATP for their metabolism. Also, as these chloroplasts do not contain ribulose 1,5bisphosphate carboxylase, photorespiration does not complicate estimation of electron transport from measurements of net 02 evolution. Material and methods Isolated mesophyll chloroplasts were prepared from glasshousegrown Zea mays L. cv. Kelvedon glory by the method of Jenkins and Russ (1984). The rate of 02 evolution, chlorophyll a fluorescence and 9-aminoacridine fluorescence were measured (Quick and Hotton 1984; Schreiber et al. 1986) in a medium containing intact chloroplasts (50 lag.ml 1 chlorophyll), 0.3 M sorbitol, 0.5 mM ethylenediaminetetraacetic acid (EDTA) 25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-KOH (pH 7.8), 3 ~tM 9-aminoacridine and 2000 U/ml catalase. The qQ and qNP components of chlorophyll a fluorescence quenching were elucidated using the light-doubling technique and the quenching coefficients determined according to Schreiber et al. 1986. Actinic illumination (520 to 660 nm, defined by a Schott [Mainz, FRG] OG 515 filter and an Ealing [Warlord, U.K.] 600 shortpass filter) was provided by a quartz projector lamp. A saturating pulse of light (2500 ~tmol quanta-m-2-s - 1, defined by the same filters as the actinic light) was flashed for a duration of 1 s every 7 s during illumination to reduce QA. This flash frequency and duration saturated QA but did not significantly affect the steady-state variable fluorescence yield or the rate of 02 evolution. Chlorophyll a fluorescence was monitored using a Hansatech (Kings Lynn, Norfolk, UK) modulated fluorescence detection system with the detection filters replaced by a Balzers (Fiirstentum, Liechtenstein) RG 715 filter. Fluorescence from 9-aminoacridine was monitored using a modulated system (Horton 1983). Oxygen was measured with a Clark-type electrode (Hansatech). All instruments were interfaced to an Amstrad (Nott,, U.K.) PC1512 micro-computer which deconvoluted the chlorophyll fluorescence quenching and differentiated the 02 signal to give a rate measurement.
Results
The rate of 0 2 evolution, photochemical and nonphotochemical components of chlorophyll fluorescence quenching (qo and qNP) and 9-aminoacridine fluorescence quenching were monitored in isolated maize mesophyll chloroplasts following illumina-
R.T. Furbank : Regulation of electron transport in maize !
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Fig. 2. Time-course of glycerate 3phosphate reduction in isolated, intact maize mesophyll chloroplasts. Glycerate 3phosphate (0.2 raM) was added in the dark with 0.5 mM Pi. After illumination, a further 0.2 pmol glycerate 3-phosphate was added (at arrow). The rate of 02 evolution ( ), qQ ( o - - e ) , qNe ( o - - o ) and 9-aminoacridine fluorescence quenching (---) are shown. The photon fluence rate in this experiment was 305 pmol. 131
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436
tion in the presence of oxaloacetate or glycerate 3-phosphate (Figs. 1, 2). After the substrate had been consumed and O2 evolution ceased, additional oxaloacetate or glycerate 3-phosphate was added. This was repeated over a 10-min time-course. Both the rate of O2 evolution and qQ increased rapidly after oxaloacetate addition, reached a maximum, then declined as the substrate was exhausted (Fig. 1 A). Qualitatively, non-photochemical quenching (qNP) and 9-aminoacridine fluorescence quenching behaved similarly, increasing with the rate of electron transport and decreasing with diminishing 02 evolution (Fig. 1 B). The kinetics of the 9-aminoacridine and qNP were, however, quite different. Non-photochemical quenching established more rapidly than 9-aminoacridine fluorescence quenching, a phenomenon which became marked during the time-course. It is unlikely that this was an instrument artifact as the response time of the 9-aminoacridine signal was less than 0.5 s while qNP was only estimated every 7 s with an instrument lag of approx. 0.3 s. It should be emphasised that while 9-aminoacridine fluorescence quenching is an indicator of thylakoid ApH, the relationship is not necessarily a linear one. Total dependence of 9-aminoacridine fluorescence quenching on the presence of oxaloacetate during illumination indicates that the majority of the ApH in these circumstances was dependent upon linear electron transport to NADP. Where glycerate 3-phosphate was used as substrate, qQ and the rate of O2 evolution behaved as in Fig. 1 but qNP and 9-aminoacridine fluorescence quenching remained high even after O2 evolution had decreased by 80% (Fig. 2). In other experiments (data not shown) where no additional glycerate 3-phosphate was added, 9-aminoacridine fluorescence quenching and qNP were maintained for up to 5 min in the absence of net O2 evolution. Thus, it appears that cyclic and pseudocyclic photophosphorylation maintained the ApH after NADP reduction ceased. Addition of glycerate 3-phosphate in the light lowered both 9-aminoacridine fluorescence quenching and qNP. Non-photochemical quenching of chlorophyll fluorescence (as measured by pulse-modulated fluorometry) is primarily a composite of energydependent quenching ( q E ) , photoinhibitory quenching (q0 and quenching caused by redistribution of energy between the photosystems (due to state transitions), termed qT (see Horton and Hague 1988). Clearly, it is important to resolve these components of qNP if meaningful correlations are to be made between qE and thylakoid ApH. The components of qNP can be resolved using
R.T. Furbank: Regulation of electron transport in maize Table 1, Relaxation times for qNP in maize mesophyll chloroplasts. The coefficient of non-photochemical quenching (qNP) was recorded after 5rain illumination ( 6 6 0 g m o l - m 2 . s - t ) with the substrates indicated (which were added prior to illumination). The tl/2 shown is the time taken for qNP to relax 50% following darkening. In a, b, and c this relaxation was complete (i.e. the F v / F m obtained after the initial illumination with a saturating flash was reached). In d, full relaxation did not occur and the initial F v / F m was never reached. The figure indicated is for the relaxation to this final value. P G A = glycerate 3-phosphate; OAA = oxaloacetate; PYR = pyruvate
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pulse-modulated fluorometry because there are differences in the relaxation times for each component (Horton and Hague 1988). Relaxation of qT and ql is quite slow (tl/2 of 8 30 min) while qE relaxes fully in less than 1 min (Horton and Hague 1988). The kinetics of this relaxation can be examined by applying a saturating flash of light following darkening of the chloroplast suspension, and recording the time-course of recovery of Fv/Fm. The frequency of flashes is chosen to be non-actinic as judged by O2 evolution and quenching of 9aminoacridine fluorescence. Table 1 shows the relaxation times for qNP in maize mesophyll chloroplasts determined by applying a series of flashes (1 s duration and at least 30 s separation) after darkening at the end of a 5-min period of illumination with various substrates, generating different values of qNP in the light. With either glycerate 3-phosphate or oxaloacetate as substrate, qNP had relaxed completely within less than 2 rain. In contrast, when no substrate was added, a component of qNP was present which did not relax completely even after 10 min darkness. In the absence of substrates, this component of qNP accounted for a large proportion of the non-photochemical quenching in the light, although the total amount of quenching was small (generally less than 20% quenching of variable fluorescence from the Fm level). From these data it appears that the majority of non-photochemical quenching shown in Figs. 1 and 2 is energy-dependent quenching (qE), although a minor contribution from the slowly relaxing component seen in Table 1 cannot be excluded.
R.T. Furbank : Regulation of electron transport in maize
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Rate of 0 2 evotufion (]Jmo[.mg-1ChLh -1) Fig. 3. Plot of photochemical quenching against the rate of O2 evolution in isolated, intact maize mesophyll chloroplasts at four photon fluence rates, 44 ( • 305 (e), 690 (z~), and 957 (A) ~tmol.m 2.s-~, with 0.1 mM oxaloacetate as substrate. These plots were generated from time-courses of the type shown in Fig. 1. Linear regression coeficients (r) were >0.85 in each case, except for the data at 44 ~tmol. m 2. s ~ for which a linear fit was considered inappropriate
Fig. 5. A comparison of the relationship between qo and the rate of 02 evolution in isolated, intact maize mesophyll chloroplasts, with oxaloacetate (0.1 mM) as substrate in the presence (A) and absence (n) of I gM antimycin A. The photon fluence rate in this experiment was 305 I~mol.m 2.s 1. The maximum values of non-photochemical quenching attained in each case were 0.3 and 0.58, respectively
Experiments of the type described in Figs. 1 and 2 were done at various photon fluence rates of investigate the relationship between the redox state of QA and the rate of 02 evolution with oxaIoacetate (Fig. 3) or glycerate 3-phosphate as substrate (Fig. 4). At moderate to high photon fluence rates, irrespective of the substrate added, qQ was linearly related to the rate of 02 evolution (as would be predicted if the redox state of QA was determined only by its rate of oxidation and a fixed
rate of electron input from PSII at a given photon fluence rate). As the photon fluence rate was increased, QA became more reduced and the slope of the relationship decreased, although linearity was maintained. At the lowest light levels examined, this relationship no longer obtained and a biphasic curve resulted. In Fig. 5, the relationship between qQ and the rate of O2 evolution was investigated with oxaloacetate as substrate in the presence of antimycin
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Fig. 4. Plot of photochemical quenching against the rate of 02 evolution in isolated, intact maize mesophyll chloroplasts at three photon fluence rates, 44 ( x ) , 305 (o) and 690 (A) g m o l - m - 2 . s -1, with 0.2 mM glycerate 3phosphate as substrate. These plots were generated from timecourses of the type shown in Fig. 2. In each case r>0.85. In the case of the data at 44 gmol. m - 2. s- t a linear fit was considered inappropriate
438
A (1 taM). Antimycin A inhibited qNP by about 50% but the plot obtained was virtually superimposable with the control (producing a linear relationship with r>0.9). Addition of NH4C1 totally collapsed 9-aminoacridine fluorescence quenching but also had little effect on this relationship (data not shown). Discussion
The relationship between qQ and the rate of 0 2 e v o l u t i o n . It is generally accepted that at "limiting" light intensities, the rate of electron transport is determined by the efficiency of photon capture, hence, this region of the light-response curve is called the "quantum yield" region. As the light intensity is increased, presumably a point is reached where the capacity for utilisation of electrons produced from water-splitting becomes limiting (either because the rate at which plastoquinol can be oxidised by the cytochrome b-f complex is too slow or simply the rate of NADP regeneration by carbon metabolism is limiting). The expected consequence of this change in limitation is the reduction of the primary acceptor of PSII, QA. It has recently been observed that this is not the case (Weis and Berry 1987; Horton and Hague 1988). Both Weis and Berry (1987) and Horton and Hague (1988) examined this effect as a function of incident light intensity with emphasis on the photo-protective nature of the phenomenon. The data shown here examines this phenomenon by manipulating the availability of electron acceptor and the magnitude of the ApH. If control of PSII by ApH is present in maize mesophyll chloroplasts, then the relationship between 02 evolution and the redox state of QA would be more dependent upon the prevailing ApH, rather than the availability of NADP. From the data of Figs. 1 and 2 there is little evidence for such control under these assay conditions. Values of qo in maize chloroplasts varied between 0.1 and 0.85 both in response to changes in NADP availability and photon fluence rate. Clearly, the relationship between qQ and O2 evolution changed with photon fluence rate (Figs. 3, 4), illustrating a decrease in total quantum yield at high light, but there appeared to be no effect of either ApH (as evidenced by 9-aminoacridine fluorescence quenching) or qyp on this relationship. Table 2 shows an attempt to compare the data of Figs. 3 and 4 by normalising o the incident photon flux. If the inverse of the slope of this relationship (i.e. electron transport/qo) is divided by the incident photon fluence rate in each plot, the pa-
R.T. Furbank: Regulation of electron transport in maize Table 2. Comparison of the data of Figs. 3 and 4 by normalising
to the incident photon flux. q5 is the rate of 02 evolution in gmol.(mg Chl)-l.h l/incident photon fluence rate in lamol. m - 2 s-1. The value qS/qo represents the photochemical yield for quanta incident on open PSII reaction centres as defined by Weis and Berry (1987). These data are taken from the slopes of the lines in Figs. 3 and 4 Photon fluence rate (lamol.m-2.s -1)
Substrate
q~/qo
44 44 305 305 690 690 957
Glycerate 3-phosphate Oxaloacetate Glycerate 3-phosphate Oxaloacetate Glycerate 3-phosphate Oxaloacetate Oxaloacetate
N.D. 0.24 0.27 0.21 0.25 0.19 0.24
rameter referred to by Weis and Berry (1987) as the "apparent quantum yield of open PSII reaction centres" is obtained. This value indicates the photochemical yield (with respect to incident quanta) of open PSII reaction centres in driving linear electron transport. Such a treatment assumes that qo accurately reflects the proportion of centres " o p e n " and that energy transfer between centres is minimal (see Weis and Berry 1987). With oxaloacetate or glycerate 3-phosphate as substrate, this value does not change appreciably over the photon-fluence-rate range 300 to 957 gmol quanta. m - 2 . s 1. At low irradiances this calculation is somewhat uncertain, however, as the relationship between 02 evolution and qo becomes non-linear. If the initial slope of the plot obtained for oxaloacetate is used in this calculation, values similar to those obtained at high light result. In the case of glycerate 3-phosphate, insufficient points are present at low rates of electron transport to make this calculation. It appears that over a small range of electron-transport rates in low light, the redox state of QA can remain constant. This occurred only at qQ values above 0.8 and could relate to the phenomenon described in intact leaves (Weis and Berry 1987). Despite large variations in the level of non-photochemical quenching and 9-aminoacridine fluorescence quenching during the time-course of each experiment (see Figs. 1, 2), the relationship between qo and the rate of 02 evolution remained linear. Antimycin A (a specific inhibitor of cyclic photophosphorylation (Moss and Bendall 1984)) has previously been shown to inhibit selectively high-energy-state quenching in thylakoids from C3 plants (Oxborough and Horton 1987). In the experiments shown here, this inhibitor abolished
R.T. Furbank: Regulationof electron transport in maize 50% of non-photochemical quenching but did not affect the relationship between q0 and the rate of O2 evolution (Fig. 5). Once again, this is in contrast with results reported in C3 leaves during steady-state photosynthesis (Weis and Berry 1987) where a linear relationship between the quantum yield of open PSII reaction centres and qsP was observed. However, it is important to remember that Weis et al. manipulated qo, qNP and the rate of O2 evolution at steady-state in leaves by adjusting the incident photon fluence rate. Thus, the rate of electron transport was varied by a change in energy input. In contrast, the experiments described here rely upon changes in the availability of N A D P as electron acceptor as substrate is supplied and then consumed. This may be analogous, in an intact leaf system, to varying photosynthetic flux by controlling the CO2 concentration, although in an intact leaf system, photorespiration is an added complication. Although it has been stated that qyp also influences the relationship between qo and Oz evolution in leaves when CO2 is changed (Weis and Berry 1987), no data have been published to date. There is, however, some evidence from studies of intact spinach chloroplasts (Furbank et al. 1987) that the relationship between qQ and electron transport may be flexible even at a given irradiance. It may be that regulation of electron transport in C4 mesophyll chloroplasts differs from that in C3 chloroplasts and a mechanism to prevent over-reduction of QA may not be present.
The relationship between qNP and 9-aminoacridine .fluorescence quenching. It appears from recent evidence that energy-dependent quenching of chlorophyll fluorescence and thylakoid ApH formation are not immutably linked (Oxborough and Horton 1987). In Figs. 1 and 2, although the behaviour of these parameters is broadly similar, the kinetic behaviour of qNP and 9-aminoacridine fluorescence quenching throughout a time-course with either glycerate 3-phosphate or oxaloacetate as substrate is somewhat different. This could be easily accounted for if a major component of qNa was unrelated to energy-dependent quenching; however, from Table 1, this appears not to be the case. Rapid relaxation of qsP in the dark under most experimental conditions (Table 1) indicates that the majority of qNP was " energy "-dependent qE-type quenching. It is unlikely that the irreversible quenching observed in the absence of substrate (possibly as a result of photoinhibition) contributed significantly to qNa with oxaloacetate or glycerate 3-phosphate present.
439 Discrepancies between the kinetics of qsP formation and ApH formation have also been observed in isolated spinach chloroplasts (Furbank etal. 1987). Although 9-aminoacridine fluorescence quenching is never seen to behave inversely with respect to qE, these observations are contrary to the close correspondence seen in other systems (Krause et al. 1982). It is also interesting to note that both 9-aminoacridine fluorescence quenching and qNa were equally affected by antimycin addition, unlike experiments with spinach chloroplasts using an artificial acceptor (Oxborough and Horton 1987). Although the rate of electron transport is linearly related to qQ in these experiments, the relationship between 02 evolution and 9-aminoacridine fluorescence quenching (and qyp) is not as clear. When oxaloacetate was supplied as substrate, with no sink for the ATP produced, the onset of 9-aminoacridine fluorescence quenching and qNP were almost entirely dependent on noncyclic electron transport (Fig. 1). In contrast, following illumination in the presence of glycerate 3-phosphate, substantial 9-aminoacridine fluorescence quenching and qsp were maintained even after net 02 evolution (and hence non-cyclic electron transport) had decreased almost to zero. This implies that the proportion of cyclic, pseudocyclic and non-cyclic electron transport are in some way regulated by the ATP demand in these chloroplasts, and that low metabolic ATP demand favours the non-cyclic pathway. Stimulation of cyclic photophosphorylation by an ATP sink has previously been proposed to explain effects of pyruvate addition on the P51s electrochromic shift in maize mesophyll chloroplasts (Crowther et al. 1983). The details of this regulation are currently under investigation. It is interesting to note that extrapolation of the data of Figs. 3 and 5 to zero 02 evolution results in a positive intercept on the qQ axis. The validity of this extrapolation is brought into question by the observation that at low irradiance, the relationship becomes curvilinear. However, if correct, it implies that low rates of electron transport out of the QApool proceed undetected during these time-courses. Photoreduction of oxygen (pseudocyclic electron transport) could be responsible for this phenomenon as net O2 exchange in this reaction in the presence of catalase is zero, rendering it undetectable by polarographic techniques. This pathway has been shown to operate under conditions where N A D P regeneration is limiting, such as the early induction phase of photosynthesis (Furbank et al. 1982) and in the absence of sub-
440
strates in C4 mesophyll chloroplasts (Furbank et al. 1983). Potential oxidation of QA by this pathway is supported by observations in spinach chloroplasts that QA is rapidly oxidised during the induction phase of photosynthesis despite the absence of detectable O2 evolution (Furbank et al. 1987). The author wishes to thank Dr. P. Horton and Professor D.A. Walker for constructive discussions.
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