Photosynthesis Research 27: 121-133, 1991. (~) 1991 KluwerAcademic Publishers. Printed in the Netherlands. Regular paper

Resolution of components of non-photochemical chlorophyll fluorescence quenching in barley leaves Robin G. Waiters & Peter Horton Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield $I0 2TN, UK Received 31 July 1990; accepted in revised form 12 November 1990

Key words: fluorescence

high-energy state quenching, photoinhibition, photosynthesis, state transition, 77K

Abstract

Non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves has been analysed by monitoring its relaxation in the dark, by applying saturating pulses of light. At least three kinetically distinct phases to qN recovery are observed, which have previously been identified (Quick and Stitt 1989) as being due to high-energy state quenching ('fast'), excitation energy redistribution due to a state transition ('medium') and photoinhibition ('slow'). However, measurements of chlorophyll fluorescence at 77 K from leaf extracts show that state transitions only occur in low light conditions, whereas the 'medium' component of qN is very large in high light. The source of that part of the 'medium' component not accounted for by a state transition is discussed.

Abbreviations: ATP - adenosine 5'-triphosphate; DCMU - 3[3,4-dichlorophenyl]-l,ldimethylurea; A p H - trans-thylakoid pH gradient; Fo, F m -room-temperature chlorophyll fluorescence yield with all reaction centres open, closed; F v -variable fluorescence = Fm-Fo; LHC I I - Light harvesting complex II; PS I, PS II - Photosystem I, II; P700, P680 - primary donor in photosystem I, II; qP - photochemical quenching of variable fluorescence; q N - non-photochemical quenching of variable fluorescence; qN e, qNt, qNi-non-photochemical quenching due to high energy state, state transition, photoinhibition; qNf, qNm, qN S- components of qN relaxing fast, medium, slow; qr - quenching of r relative to the dark state; tricine- N-tris[hydroxymethyl]methylglycine; r - ratio of fluorescence maximum from photosystem II to that from photosystem I at 77 K Introduction

Chlorophyll a fluorescence gives a direct measure of the level of excitation of a photosynthetic pigment system; a reduction in chlorophyll excitation due to an increase either in utilisation via photochemistry or in the dissipation of energy as heat is accompanied by quenching of the fluorescence yield. At room temperature, fluorescence is derived predominantly from Photosystem II, and therefore provides a powerful non-intrusive means of investigating the mechanisms whereby

thylakoids regulate the utilisation of absorbed light energy by PS II. Increased light utilisation (photochemistry) or energy dissipation lead to quenching of the fluorescence yield to levels below the maximum; however, proper interpretation of such information relies on the ability to quantify the contributions to total fluorescence quenching made by PS II photochemistry (qP) and by each of several other, distinct, nonphotochemical processes (qN). The identification of factors involved in qN in aqueous photosynthetic systems has been aided

122 by the use of inhibitors and uncouplers (Briantais et al. 1979, Horton et al. 1987, Horton and Hague 1988): DCMU inhibits electron transport away from photosystem II, removing photochemical quenching and thus revealing the extent of non-photochemical quenching; uncouplers such as nigericin and NHaCI dissipate the transthylakoid ApH, eliminating high-energy statedependent dissipation (qN~); sodium fluoride inhibits the phosphatase responsible for reversing LHC II phosphorylation, which is believed to be associated with the redistribution of excitation energy away from photosystem II (qNt); and recovery of photoinhibitory damage (qNi) is blocked by inhibitors of de novo protein synthesis. However, when studying intact leaf systems, inhibitors are of limited use, and other approaches must be devised. Other forms of nonphotochemical quenching by species such as oxidised plastoquinone, reduced pheophytin, oxidised P680 and oxidised P700 are more difficult to quantify and it is unclear whether they are important in vivo (for a review see Baker and Horton 1987). The description of the 'light-doubling' technique (Bradbury and Baker 1984) enabled measurement of the contribution of Photosystem II photochemistry to fluorescence quenching using rapid changes in the incident light intensity. The application of brief pulses of intense light transiently closes all PS II centres and removes photochemical quenching; any remaining fluorescence quenching is (by definition) non-photochemical in nature. In this way, Horton and Hague (1988) monitored the relaxation of non-photochemical quenching in barley protoplasts returned to the dark after a period of illumination, by periodically applying pulses of saturating light, qN relaxed in three approximately exponential phases, decaying with half-times of 30 s, 8 min and 30 min; these decay times correlate well with those expected for qNe, qN t and qN i. Furthermore, the quenching coefficients attributed to each phase of recovery were comparable to those obtained using inhibitors, suggesting that there may be causal relationships between the phases of qN recovery and the relaxation of specific mechanisms of non-photochemical quenching. If this were true, such an approach could provide a means of deconvoluting qN in intact leaves.

Following the work of Demmig and Winter (1988), who used saturating pulses to separate qN in intact leaves into relaxing and non-relaxing components, Quick and Stitt (1989) have analysed the kinetics of qN relaxation in barley leaves. As with protoplasts, three phases were observed with properties similar to those expected for components of qN: infiltrating tentoxin or nigericin into leaves via the transpiration stream increased or decreased the amplitude of the 'fast' phase (with a half-time of 1 min), consistent with it being due to the relaxation of qN e; and treatment with fluoride (to inhibit dephosphorylation of LHC II, thereby preventing relaxation of a state transition) reduced the size of the 'middle' phase (with a half-time of 5 min), consistent with it being associated with qN t relaxation. It was found that this measure of qN t rose steadily in increasing actinic light, reaching a plateau in near-saturating light. However, one might predict that state 1-state 2 transitions would become saturated in low light, since it is in such conditions that energy distribution between photosystems would limit photosynthesis. Indeed, it has been shown in maize mesophyll chloroplasts that the level of LHC II phosphorylation is reduced in strong light (Fernyhough et al. 1984). Furthermore, the quenching associated with the middle phase measured at high light, with a coefficient greater than 0.4, was much greater than any previously reported qN t (Horton and Black 1981, Horton and Hague 1988). Two simple explanations for the apparent inconsistencies between qN relaxation and in vitro data present themselves: state transitions may occur in intact leaves with a different lightdependency compared to that observed in aqueous systems; or analysis of the kinetics of qN relaxation may not be an accurate means of quantifying the different quenching processes comprising qN in leaves, perhaps because part of qN e relaxes more slowly than in vitro. In Dunaliella cells a component of qN e has been found to persist for several minutes after darkening (Lee et al. 1990). In an attempt to discriminate between these possibilities, we have deconvoluted qN in barley leaves over a range of light intensities in a manner similar to that employed by Quick and Stitt (1989), reaching qualitatively similar conclusions. The quenching associated

123 -2

Chlorophyll fluorescence and oxygen measurements Chlorophyll a fluorescence yield and oxygen evolution at 20°C in saturating CO 2 was measured simultaneously during and after illumination using a modified Hansatech LD2 leaf disc chamber and a Walz PAM101 fluorimeter. The weak modulated measuring beam (1.6 kHz, increased to 100 kHz during illumination) had no effect on the dark-adapted Fv/F m ratio. Actinic light was from a Schott KL1500 lamp fitted with a KG1 heat filter and a no. 511 conversion filter, which provided illumination enriched in PS II light in comparison with that experienced by the plants during growth (see Fig. 1). Far-red light (740 nm, bandwidth 10 nm) was supplied using an Ealing interference filter. Pulses of saturating light (3000 ~ E m -2 s -1) were delivered from a heat-filtered General Electric ELC projection lamp via an electronic shutter controlled from a Walz PAM103 Trigger Control unit. Fluorescence yield and oxygen data were recorded on an Opus PCV computer using software developed by J. McAuley and J.D. Scholes (University of

Materials and methods

Plant material Barley was grown from seed at 20-25°C under metal-halide lamps supplemented with tungsten filament lamps and heat-filtered through glass tanks of water, at an intensity of 250-300/xE -2 ] m s - ; seven day-old seedlings were darkadapted for 18-24 h before use. Leaf samples approx. 8 mm long were taken 25 mm from the tip; where necessary they were treated by stripping the epidermis from the back and floating them on 20 mM NaF (or on water for a control), firstly for 1 h under weak illumination (70/xE

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with the 'middle' phase of qN recovery in barley leaves has then been compared with the quenching of PS II fluorescence due to a state transition, identified by measuring chlorophyll fluorescence at 77K from extracts of identically-treated leaves. This measure of the extent of a state transition gives results markedly different from those obtained using qN relaxation kinetics. We conclude that the middle phase of qN relaxation contains contributions from both state transitions and high-energy state quenching, although at low light intensities that from the state transition predominates.

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124 Sheffield). The fluorescence quenching parameters qP and qN were calculated using the quenched F o level as described by Quick and Stitt (1989); those associated with components of steady-state qN (distinguished on the basis of their relaxation kinetics: q N f = f a s t , qN~,= medium, qN s = slow/irreversible) were derived by extrapolating back semi-logarithmic plots of qN versus time, applying the relationship (1 - qN) = (1 - qNf) × (1 - qNm) x (1 - qNs) (see below).

than 2 min, during which time, in the presence of nigericin, all ApH-dependent fluorescence quenching (qNe) relaxed. At room-temperature, the F v / F m ratios of extracts from dark-adapted leaves (0.78) were comparable with those for intact leaves (0.81), demonstrating that the extraction procedure left the photosynthetic apparatus largely intact. Samples were illuminated using modulated 460 nm light (20 nm bandwidth, approx. 35/~E m -2 s -a) and fluorescence spectra were recorded using a scanning monochromator, photomultiplier and lock-in amplifier. Fluorescence peaks at 685 nm (PS II) and 735 nm (PS I) were normalised against chlorophyll concentration and the constant 545 nm fluorescein reference peak. Samples were scanned at least three times in different orientations, to avoid errors due to extract heterogeneity. All error bars show the standard errors from at least three extracts.

Low-temperature fluorescence measurements 77 K fluorescence in leaf extracts was met oured as follows. Immediately after illumination, the leaf sample was ground in ice-cold extraction medium (0.4M sorbitol, 10mM tricine-HCl pH8.4, 50mM MgC12, 50mM NaF, 10/xM nigericin) with a mortar and pestle and was filtered through a cotton wool plug in a Pasteur pipette. Chlorophyll concentrations of filtered extracts were measured spectrophotometrically as described by Arnon (1949). 125/zl of extract was added to 25 ~1 10/xM fluorescein before freezing in liquid nitrogen; this process took less

Results

Relaxation of qN in the dark Figure 2 shows the fluorescence yield and oxygen evolution typical of a dark-adapted leaf illumi-

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Time/mins Fig. 2. Chlorophyll fluorescence yield and rate of oxygen evolution from a barley leaf segment during illumination and dark recovery. The leaf segment was illuminated for 20 min (900/.~E m -2 s - ) and redarkened. A 3 s pulse of saturating light (3000/xE m -2 s -2) was given 2 min prior to darkening to allow measurement of the steady-state qP and qN, and 1 s pulses were given 5 s after darkening and every 120 s thereafter to enable qN relaxation to be monitored (see text).

125 nated for 20min (900 ~ E m -2 s -~) and then returned to the dark (apart from the 1.6kHz measuring beam). On illumination, the initial fluorescence maximum is rapidly quenched, reaching a steady state for oxygen evolution and fluorescence quenching within 20min. A 3s pulse was given 2min prior to darkening to enable measurement of the steady-state qP and qN; that qN and qP were at steady state was demonstrated by repetitive saturating pulses during the light period (not shown). Immediately after the 3 s pulse, fluorescence is transiently quenched to below the steady-state level, possibly because of additional qNe quenching resulting from a temporarily increased ApH. After darkening, there is a rapid decrease in fluorescence as PS II centres become open, to a level substantially below the dark-adapted level. Nonphotochemical fluorescence quenching during recovery in the dark was periodically monitored by the application of pulses of high-intensity light (3000 k~E m -2 s -I) of 1 s duration 5 s after darkening and every 120 s thereafter. The first saturation pulse, given 5 s after darkening, gives a fluorescence peak lower than that obtained immediately before darkening, and was ignored in the analysis of recovery data; Quick and Stitt (1989) ascribed this to an inability of strong light to fully close all PS II centres in this state. Subsequent pulses revealed qN to be relaxing in a complex manner. The repetitive pulses applied during dark recovery might affect the relaxation of qN, so it was important to ensure that the strength, length and frequency of the saturating pulses allowed complete relaxation back to the dark state, while providing the maximum information about dark recovery. Quick and Stitt (1989) have carried out a systematic analysis of the effect of pulse strength and frequency on qN recovery in barley; they concluded that qN relaxation was largely unaffected by light pulse regimes which caused little change in the 'dark' level F v / F m ratio. The pulse regime used above depresses the Fv/F m ratio by no more than 2% when applied to dark-adapted leaves, and allows complete recovery of leaves given low light. Three distinct exponentially decaying phases to the relaxation of qN can be observed. The relaxation half-times of each phase (60s, 5-

10 min and >30 min: see below) are similar to those observed for qN e (previously termed qE), qN t (qT) and qN i (qI) in barley protoplasts (Horton and Hague 1988), and in barley leaves (Quick and Stitt 1989). We here use a modified form of the nomenclature used by Quick and Stitt for the quenching coefficients for each phase of recovery: fast-relaxing quenching is termed qNe; similarly q N m and qNs describe quenching corresponding to the 'middle' and 'slow' phases of qN recovery.

Deconvolution of qN relaxation kinetics Quick and Stitt (1989) analysed their dark relaxation data by linear extrapolation of each phase of recovery back to the time of darkening and applying the relationship (1 - qN) = (1 - qNf) x (1 - qN~) x (1 - qNs). This method of calculation is only accurate as long as the components of qN relax linearly; however, our data show a much better fit if exponential decay is assumed. Figure 3 illustrates the deconvolution of dark recovery data into exponentially decaying components for several leaves illuminated with 950/xE m -2 s-~: using semi-logarithmic plots of qN versus recovery time, fluorescence quenching parameters associated with each phase were calculated by linear extrapolation as shown; after determination of the qN S component and its decay half-time, the data were recalculated in order to remove that component; the transformed data were treated in the same way to derive qNm, and then qNf.

Actinic light-dependency of components of qN The effects of actinic light intensity on steadystate chlorophyll fluorescence quenching, components of qN and the rate of oxygen evolution in the steady state are shown in Fig. 4; qN deconvolution was by the method described above. Other methodologies including that used by Quick and Stitt (1989) gave qualitatively similar but quantitatively different results (not shown). Oxygen evolution increases linearly with light intensity up to approx. 150/xE m -2 s-1, but as the acceptor side of photosystem II becomes progressively reduced in higher light, electron transport becomes saturated and a sharp drop in

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Recovery Time /mins Fig. 3. Deconvolution of qN recovery data from barley leaves illuminated with 900 ~tE m -2 s -2. The magnitude and decay half-time of the 'slow' component of recovery was measured by linear regression through the latter (linear) part of a semi-logarithmic plot of raw recovery data (left-hand panel). These data were transformed (see text) to give a second set of data from which the 'medium' component was measured (middle panel). Repeating the procedure gave the 'fast' component (right-hand panel).

qP is observed; there is relatively little change in qP where oxygen evolution is light-limited. In contrast, there is significant non-photochemical quenching even under very weak illumination, increasing in limiting light and yet further in high light. The two data points seen at approx. 270/zE m - 2 s - 1 represent two distinct groups of leaves separated on the basis of their relaxation kinetics from light intensities in the range 240-280 ~ E m -2 s -1. This may reflect a sharp transition in the manner in which the plant reacts to light, from one where light is regarded as limiting to one where it is in excess; the light intensity at which this occurs corresponds precisely with the principal inflection point on the oxygen evolution curve, and also with the light intensity at which the plants were grown. Alternatively, there may be two sub-populations of leaf which behave slightly differently at this light intensity. However, this was the only set of data where such a wide spread of values for qN and qP were observed, and where qN relaxation was highly variable. We therefore favour the former interpretation. The individual components of qN show lightdependencies qualitatively similar to those reported by Quick and Stitt (1989). In low light,

qN consists almost entirely of q N m , which remains fairly constant up to the point at which oxygen evolution begins to become saturated; over this intensity range there is very little contribution from either qNf or qN s. In non-limiting light, all three components of qN show a sharp increase, qNf and qN m rapidly rising to plateaux at approx. 0.4 and 0.6, respectively, qN s increasing throughout the light intensity range, qO, quenching of the F o level of fluorescence, rises roughly in parallel with qNf, but without the sharp increase seen at around 240-280/zE m - 2 s-1.

The time constants for components of qN (Fig. 5) do not remain constant across the entire light-intensity range. There is an abrupt increase of approx. 50% in the decay half-time of qNf after illumination with high light, perhaps suggesting that the ApH is dissipated more slowly. There is also an increase in the recovery time for qNs in high light-treated leaves, perhaps reflecting the presence of an additional quenching process within this component of qN; the capacity of the available protective (fluorescence quenching) mechanisms may have been exceeded, leading to irreversible damage and the observed rise in the decay half-time of the slow phase of qN recovery. In contrast, the decay half-time of q N m

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shows a marked decrease over a range of much lower light intensities, but is constant in high light; this suggests either that the mechanism associated with qN m relaxes at a faster rate after high light, or that two different forms of quench-

ing with different decay times comprise q N m , one being present in low light but being gradually replaced by the second in increasing light.

Effect of fluoride on fluorescence quenching and its recovery Sodium fluoride inhibits the relaxation of a state 1 to state 2 transition by preventing dephosphorylation of LHC II (Canaani et al. 1984). Treatment of leaves with fluoride to fix them in state 2 should therefore modify the relaxation of non-photochemical quenching, since state transitions will not occur, qN relaxation in fluoridetreated leaves was monitored and deconvoluted as described above at two light intensities; the results are shown in Table 1. The results for 'water-treated' leaves were indistinguishable from those for intact leaves.

Table 1. Fluorescence quenching coefficients in fluoride-treated and control leaves at two light intensities. Leaves floated on water or 20 mM NaF were illuminated (70/zE m z s-l) for 1 h and then redarkened prior to analysis of qN relaxation (see text). The relaxation half-times of components of qN (in minutes) are shown in brackets Light intensity

70/zE m -2 s -1

900 p~E m 2 s -

Treatment

NaF

water

NaF

water

qP qN qN qN m qNf

0.92 0.42 0.09 (28) 0.05 (5.0) 0.33 (1.3)

0.95 0.23 0.04 (50) 0.14 (8.3) 0.07 (0.9)

0.12 0.89 0.42 (157) 0.31 (4.2) 0.72 (1.3)

0.37 0.87 0.47 (194) 0.58 (5.0) 0.41 (1.2)

128 The effects of fluoride on fluorescence quenching are complex, possibly because it causes a reduction in the light-saturated rate of photosynthesis (not shown) in addition to its effects on protein dephosphorylation. As a result, qP in high light is markedly lower while qN is largely unchanged; in contrast, qN is markedly increased in low light, as a result of the formation of a substantial fast-relaxing form of quenching. As expected, qN m is reduced in low light, but a substantial q N m is still seen in high light, suggesting that at least part of the middle phase of qN relaxation is due to a fluoride-insensitive process (i.e., not a state transition); it is noticeable that the qN m observed after low light has an altered decay half-time, qN s is unaffected by fluoride.

Low-temperature fluorescence from leaf extracts It is well documented that unless care is taken to dilute photosynthetic membranes, low-temperature fluorescence can be subject to artefacts due to differential re-absorption of the emitted light (Weis 1985). In order to establish the correct extent of dilution, PSI and PS II fluorescence from samples from dark-adapted leaves was measured over a range of chlorophyll concentrations (Fig. 6). Absolute measurements of PSI -1.5 -1.4 ,~ -1.3 -1.2 -..~ -1.1

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Fig. 6. Titration of 77 K chlorophyll fluorescence vs. chlorophyll concentration. Dilutions of extracts of dark-adapted barley leaves were cooled to 77 K, excited with 460 nm light (bandwidth 20 nm) and their emission maxima at 685 nm and 735 nm measured. PS I and PS II signals are normalised against the 545 nm fluorescence maximum from a constant amount of fluorescein; the ratio of the two signals was calculated for individual samples prior to averaging. Each point is the mean of 3-6 samples; error bars show the standard error (for fluorescence signals) or standard deviation (for chlorophyll concentrations).

and PS II fluorescence per unit chlorophyll are subject to wide variation, presumably due to heterogeneity between leaf extracts (standard errors for individual extracts were very small); however, the PS II/PS I ratio provides a relatively error-free means of assessing the relative proportions of absorbed light energy available to the two photosystems. It is clear that, in our apparatus, reabsorption artefacts in extracts of barley leaves are negligible at chlorophyll concentrations below approx. 10/xg ml-1; all other data are from extracts with chlorophyll concentrations below this level. Far-red light is known to lead to establishment of state 1, apparently via its effects on the redox state of the plastoquinone pool (Williams and Allen 1987). In contrast, sodium fluoride 'fixes' leaves in state 2. The presence of a state 1 to state 2 transition should therefore be revealed if, after illumination with weak light and re-darkening, fluoride-treated leaves are compared with untreated or far-red illuminated leaves. Any state transition occurring under low light will relax in control leaves, but not in those treated with fluoride (Canaani et al. 1984). Control leaves gave identical results to those from intact, dark-adapted leaves. Table 2 shows the ratios for PSII to PSI fluorescence at low-temperature in extracts from leaves treated as above, and also from leaves given other light treatments, including the light pulse regime used to monitor qN recovery. As expected, far-red light leads to increased PS II fluorescence relative to PS I, while fluoride treatment decreases it, thus demonstrating that 77 K fluorescence is a practicable method for assaying for state transitions in barley. The 1.6kHz measuring beam and the recovery regime appear to be approximately neutral, but more frequent pulses (one pulse every 10s) induces a very strong transition to state 2; this regime is similar to one used by Quick and Stitt to prevent relaxation of the middle phase of qN relaxation. The efficient state transition induced by such a light treatment may be a result of a combination of a high level of plastoquinone reduction and a small ApH; it has been suggested that a high energy state may be antagonistic to the formation of a state transition (Fernyhough et al. 1984, Oxborough et al. 1987).

129 Table 2. 77 K fluorescence (in arbitrary units) from P S I and PS II in extracts from barley leaves per unit chlorophyll, normalised against fluorescence at 545 n m from fluorescein. Control and fluoride-treated leaves had previously been exposed to 7 0 / z E m -2 s -1 light for 1 h and redarkened Treatment

PSI

Control Far-red light Fluoride Measuring b e a m Light pulses: 0.0083 Hz 0.1 Hz

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To assess the dependence of state transitions on light intensity, low temperature fluorescence was measured from extracts previously illuminated at a range of light intensities in conditions identical to those used for the analysis of qN recovery; the resulting PS II/PS I ratios are shown in Fig. 7. Although at high light intensities there is substantial ApH-dependent quenching, such quenching is efficiently removed during extraction and prior to freezing; when nigericin was omitted from the extraction medium, the PS II signal was greatly depressed relative to PSI in extracts of leaves illuminated with approx. 900/zE m -2 s(see Fig. 7). These low temperature fluorescence data therefore represent only quenching which is ApH-independent. 1.55

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Fig. 7. Light intensity-dependence of excitation distribution between PS II and PSI. Extracts of barley leaves illuminated for 2 0 m i n were treated as in Fig. 6 and the P S I I / P S I fluorescence ratio m e a s u r e d for those with final chlorophyll concentrations of less than 10/xg m1-2 (open symbols). Extracts were also prepared from leaves subject to 900/~E m -2 s -2, omitting nigericin from the extraction buffer (closed symbol).

1.480 1.585 1.371 1.465

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In leaves illuminated even at the lowest light intensity used (8.5/~E m -2 s -1) there is a marked change in the distribution of light energy between PS I and PS II, but under increasing illumination the PS II/PS I ratio rapidly reaches a minimum and then increases again, returning in saturating light to the value seen in the dark. At very high light intensities a gradual decline in the PS II/PS I ratio is once again observed; it seems probable that the source of this change is a photoinhibitory process observed as qN s. Figure 8 shows how quenching of the PS II/ PSI ratio r (calculated as qr = 1 - rnew/rdark) varies with the total non-photochemical quenching under these experimental conditions, with qN m shown for comparison. At low light (and hence low qN), the relationship between qN and qr appears to be linear, confirming that non-photochemical quenching is almost entirely due to a state 1-state 2 transition in such conditions. Furthermore, the line-of-best-fit derived by leastsquares regression passes through the origin and has a slope of 1.00 -+ 0.07, implying that qN, and qr are numerically equal; quenching due to a state transition (qNt) can therefore be estimated directly as the quenching of the PS II/PS I ratio. This observation would suggest that quenching of PS II fluorescence due to a state transition is not accompanied by redirection of energy towards PS I, since this would be expected to result in a gradient substantially greater than unity. The use of qr in the estimation of qN t is likely to be incorrect only for high values of qN (i.e., under high light), when qN t is probably zero with qr being due to photoinhibition; for barley this is the case where qN rises above approx. 0.85. The extent of the reduction in the PS II/PS I

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0.9

,, 1.0

total non-photochemical quenching Fig. 8. Standard curve to enable estimation of excitation redistribution from the total non-photochemical quench. Steady-state non-photochemical quenching (qN) was measured for illuminated barley leaves, from which extracts were made and their PS II/PS I fluorescence ratio at 77 K measured. Quenching of this ratio (q,) is shown as a function of qN (closed circles); each point comprises 3-6 separate leaf extracts. The individual measurements comprising the first data point are also shown (open circles); the broken line shows the result of linear regression through these points. For comparison, the relationship between qN and the medium component of qN relaxation (qN,,) is also shown (triangles).

ratio in very high light is substantially less than might be expected from the qN relaxation data, which give high values for qN s in high light. Two possible explanations present themselves: that the majority of the slowly relaxing form of qN is due to a mechanism which does not quench fluorescence at low temperature; or that for intact leaves, room-temperature fluorescence (and photoinhibition) is derived from (and detected at) the leaf surface, whereas an extract is of a whole leaf segment, so that any observed quenching is diluted. When qN was measured from the underside of a strongly illuminated leaf (1200/xE m -2 s -1) and compared with that on the topside, it was found to be significantly lower, implying that there is such a 'sampling artefact'. Nevertheless, qN was still at a level at which a significant slow-relaxing component was present. Further, the F v / F m ratio of an unfrozen extract from such leaves (0.71) compared to that from dark-adapted leaves (0.78) indicated the presence of ApH-independent quenching with a coefficient of approx. 0.25, substantially greater than the quenching seen at 77 K.

Discussion

An analysis of the relaxation of non-photochemical chlorophyll fluorescence quenching in barley

leaves after redarkening has revealed three distinct phases of recovery, in agreement with previous studies (Demmig and Winter 1988, Quick and Stitt 1989). These phases are separated on the basis of their decay kinetics, giving associated quenching coefficients (here termed qNs, q N m and qN~), which show varying light-intensity dependences. Although qualitatively similar, our data show differences from those of Quick and Stitt (1989) with respect to the relative sizes of the three components, the most important reasons for which are probably the different methods used for deconvolution of qN and the use of different actinic light sources. We have also measured fluorescence at 77 K from PS II and PS I, to test for redistribution of absorbed light energy between the two photosystems and for other ApH-independent quenching mechanisms. A comparison of leaves subject to state 2 conditions with leaves in state 1 shows that fluorescence from PS II relative to that from PSI decreases in state 2, as expected. The middle phase of qN recovery has previously been associated with the relaxation of a state transition. However, it is clear from the 77 K fluorescence data that while this may be true in low light, the relationship breaks down in high light (see Fig. 8); in view of this, some other quenching process must be responsible for at least part of q N m in high light. This would be consistent

131 with the observed variation in the decay halftime of qNm, from 10min after low light to approx. 5 min either after high light or after treatment with fluoride. Further evidence that the qN m formed at high light is not due to a state transition comes from 77 K fluorescence from extracts of leaves during recovery from low and high light: after low illumination, the PS II/PS I ratio rapidly recovers to near the dark value; after high light, the PS II/PS I ratio is virtually constant over the following 40 min (unpublished results). The assigning of the very large qN m formed under high light to a state transition by Quick and Stitt (1989) depended on the partial inhibition of its relaxation by fluoride. However, the comparison of control and fluoride-treated leaves was made using a two-stage relaxation, first giving saturating pulses every 7 s and allowing the leaves to reach a steady state, and then using the normal relaxation pulse regime (pulses every 100 s); it was intended that the fast pulses would allow relaxation of the fast component of qN but not of subsequent components. However, the data may be explained if a state transition is formed during the first stage; when we gave leaves saturating pulses every 10 s (the larger interval was to take account of the longer and stronger pulses used in our experiments), 77 K fluorescence showed a strong transition to state 2. When considering the qN S component, both quenching coefficient and decay half-time are relatively unaffected by deconvolution method (unpublished observations). This component is associated with photoinhibition in vitro, since its relaxation requires de novo protein synthesis (Horton and Hague 1988), yet results in quenching of the PS II signal at 77 K which is only a fraction of that seen at room temperature, even when potential errors due to varying illumination through the leaf are taken into account. When PS II fluorescence quenching is seen at 77 K, the decay half-time of qNs is much increased, suggesting the involvement of two processes in qN s: one which quenches fluorescence at room temperature, relaxes with a half-time of 30-60 min and does not lead to quenching at low-temperature; and in very high light, a second process which is slowly relaxing or irreversible and which

does quench at 77 K. The former may well be due to a readily-reversible limitation on electron transport on the donor side of PS II; for example, cross-linking of tyrosine-161 of the D1 protein (believed to be 'Z': Metz et al. 1989) and another tyrosine residue, as reported by Ohad et al. (1990), would be consistent with a donor-side limitation. The latter may be due to photooxidative damage, or perhaps a second, irreversible stage in photoinhibition. Bradbury and Baker (1986) have noted that photoinhibition appears to be a two-step process, since roomtemperature fluorescence is quenched more effectively than 77K fluorescence under mild photoinhibitory conditions. Quick and Stitt (1989) reported that qNf could be modified by manipulating the trans-thylakoid ApH with nigericin or tentoxin, implying that it is due to ApH-dependent high-energy state quenching (qNe). There is evidence to suggest that when qN m is not accounted for by a state transition, it too is likely to be associated with qNe: the high-light form of qN m quenching is not observed at 77 K in the presence of nigericin; and qNf saturates at levels substantially lower than those seen for qNe in a wide range of aqueous systems (Horton and Hague 1988, Laasch and Weis 1989, Lee et al. 1990). We would propose that in high-light, something prevents rapid relaxation of high-energy state quenching; a substantial delay in the relaxation of ApH-dependent quenching has been observed in Dunaliella (Lee et al. 1990). This could be a passive process, for instance where the metabolic requirement for ATP is such that the ApH dissipates slowly; the observed increase in the decay half-time of qNf would be consistent with such a hypothesis. Alternatively, PS II and/or its associated light-harvesting proteins might be modified in such a way that high-energy state quenching persists at a much lower ApH, a role suggested for the zeaxanthin formed in a variety of plants and algae when they are subject to high irradiation (Rees et al. 1989, Adams et al. 1990, Demmig-Adams et al. 1990, Noctor et al. 1991). It has been proposed that protein phosphorylation and zeaxanthin formation may be antagonistic processes (Horton 1989). It is hence significant that fluoride-treated leaves show a reduced qN m and an increased qNf in high light;

132 this may be because zeaxanthin formation is inhibited in such leaves, which have been 'fixed' in state 2 and therefore have high levels of irreversible protein phosphorylation even in the dark. The increase in qNf observed in low light in fluoride-treated leaves may be due to a larger ApH generated by greater cyclic electron transport around PS I, as a result of fixing leaves in state 2. It is clear that it is inappropriate to attempt to deconvolute qN by an analysis of the dark relaxation kinetics alone. Although the slowly relaxing portion of qN probably provides an accurate measure of quenching due to photoinhibitory processes (qNi), the phase relaxing with a halftime of 5-10 min seems to contain contributions from both a state transition and high-energy state quenching, resulting in unreliable measurement of both qN e and q N t. A comparison of the results of Quick and Stitt (1989) with those above illustrate how small changes in methodology can have large effects on the calculated quenching parameters; in any case, the low-temperature fluorescence data demonstrate how inaccurate such a method can be. However, 77 K fluorescence provides a means of assaying the quenching due to a state transition; a corrected value for qNe may therefore be calculated as the remaining quenching. In a subsequent paper, we shall use this combination of qN relaxation and low-temperature fluorescence to quantitatively separate components of qN, allowing for the first time an accurate analysis of theoretical models describing possible mechanisms behind chlorophyll fluorescence quenching.

Acknowledgements We are grateful to Dr P. Quick and Prof. M. Stitt for providing data prior to publication, and also for various unpublished results. This work was supported by a grant from the AFRC.

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