Effects of incident light intensity on the yield of steady-state chlorophyll fluorescence in intact leaves. An example of bioenergetic homeostasis

l'2nri~oltrnental and Experimental Botanv, Vol. 3 l, No. 1, pp. 23 32, 1991

0098 8472/91 $3.00 + 0.00 ~, 1991 Pergamon Press pit:

Printed in Great Britain.

EFFECTS OF I N C I D E N T L I G H T I N T E N S I T Y O N THE YIELD OF S T E A D Y - S T A T E C H L O R O P H Y L L F L U O R E S C E N C E IN I N T A C T LEAVES. AN E X A M P L E OF B I O E N E R G E T I C H O M E O S T A S I S M I C H E L H A V A U X , * ~ ++ R E T O

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STRASSER* and H U B E R T G R E P P I N ~

* Universit~ de Gen~ve, Laboratoire de Bio~nerg~tique, Station de Botanique, C,H-1254 Lullier-Gen~ve, Switzerland and ~"Laboratoire de Physiologic et Biochimie v6g~tales, 3 place de l'Universit6, CH-1211 Genave 4, Switzerland

(Received 23 February 1990; accepted in revisedform 14 July 1990) HAVAUX M., STRASSERR. J. and GREPVrN H. Effects of incident light intensity on the yield of steadystate chlorophyll.fluorescence in intact leaves. An example of bioenergetic homeostasis. ENVmONMENTALAND EXPERIMENTAL BOTANY 31, 23-32, 1991. The emission of modulated 685-nm chlorophyll fluorescence from intact leaves was measured using a pulsed (yellow) exciting light of very low intensity combined with various non-modulated lights of sub- and supersaturating intensities. It was found that, whereas the "extreme" (initial and maximal) fluorescence levels were drastically quenched by high light intensities, the level of steady-state modulated fluorescence in lightadapted leaves was virtually, constant and independent of ttle intensity and the quality of the continuous light used to drive photosynthesis. These results provided an example of biological homeostasis. For instance, the modulated fluorescence emission from a dark-adapted leaf with all photosystem II reaction centers in the open configuration was similar to the steady-state modulated fluorescence emission measured in leaves illuminated with an additional strong, photosynthetically saturating light with all photosystem II traps closed. This homeostatic behavior was characteristic of only healthy and mature leaves. It was strongly perturbed in young leaves and also in leaves exposed to unfavorable environmental conditions. In some stress situations (e.g. leai'dehydration), noticeable deviations t?om homeostasis were detected befbre any significant changes in the familiar photochemical (qp) and non-photochenrical (qNe) fluorescence quenching coefficients could be observed, suggesting that the analysis of this "bioenergetic homeostasis", revealed by the examination of raw fluorescence data, could be a useful approach tbr early detection of subtle changes in the physiological state of plants.

+ To whom all correspondence should be addressed at: Universit6 de Gen~ve, Laboratoire de Bio~ncrg6tique, Station de Botanique, CH-1254 Lullier-Genave, Switzerland. Abbreviations: Chl, chlorophyll; l~,d and b~,l,basic (or initial) Chl fluorescence level in dark- and light-adapted leaves, respectively; F,l,d and F,,I~, maximal Chl fluorescence level in dark- and light-adapted leaves, respectively; Fs, steady-state Chl fluorescence level; Fc,d,, basic Chl fluorescence level observed after short-term adaptation (around 30 sec) to the dark of leaves previously adapted to light conditions; qe and qNe, so-called photochemical and non-photochemical Chl fluorescence quenching coefficients; Rs, Rm~, Ro~, Ro~,, Chl fluorescence ratios calculated with the formula Rx = (Fx-Fo~)/(F,~ld-F,,d) with Fx = bs, F,,,I, F,,~ or F,,~,; QA, primary (stable) electron acceptor ofPS II; LA, actinic blue light; LVR, far-red light; PS, photosystem. 23

24

M. HAVAUX et al. INTRODUCTION

A PART of the light energy absorbed by photosynthetic pigments in green plants is re-emitted as fluorescence. At physiological temperatures, variable fluorescence emission measured in leaves predominantly emanates from chlorophylls (Chls) of photosystem (PS) II, with maximal emission occurring at around 685 nm. The intensity of in vivo PS II-Chl fluorescence is known to be affected by numerous photochemical and nonphotochemical processes including oxidation/ reduction of the primary quinone electron acceptor of PS II (QA), the light-induced p H gradient across the thylakoid membranes and the state I state II transitions. !4) Until recently, useful information concerning photosynthesis was essentially derived from Chl fluorescence induction curves obtained during dark-to-light transitions. When pre-darkened leaves are suddenly illuminated, complex changes in the Chl fluorescence yield occur and are known as the Kautsky effect, (19) reflecting sequential light activation of the photosynthetic processes mentioned above. After prolonged irradiation, leaves attain a state characterized by a low Chl fluorescence emission.(2'l~'~9) The recent introduction of selective modulation fluorometers (3/ has fostered considerable interest in the use of steady-state fluorescence measurements performed in fightadapted leaves. By combining modulated and non-modulated lights, it is possible to monitor, under any steady-state condition, the extreme (initial and maximal) levels of chlorophyll fluorescence together with the actual fluorescence level and to calculate, from those levels, various parameters such as the so-called photochemical (ql,) and non-photochemical (qNe) fluorescence quenching components.!2) Quenching analysis of steady-state Chl fluorescence emission has been used to examine photosynthetic electron transport rates and their regulation in vivo, (11'17'24''27) a s well as to investigate stress effects on leaf photosynthesis (see, for example, Refs 15 and 25). A modulated fluorescence approach was used in the present paper. A careful analysis of the (low) steady-state emission of in vivo modulated Chl fluorescence in leaves exposed to a large range of light intensities (from zero to saturation) indicated that a plant leaf can be considered as a

system in homeostasis as regards its efficiency of radiative energy dissipation, since the intensity of steady-state modulated fluorescence was the same for all the actinic light intensities tested. This conclusion appears to be valid, however, only for non-stressed and well-developed leaves. MATERIALS AND M E T H O D S

Plant material Intact leaves of pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) were used. Plants were grown from seeds in small pots filled with compost at a temperature of 20__+2°C. Pea was grown in a glasshouse under sunlight conditions and spinach was grown in growth chambers under controlled artificial light conditions. Cold stress was induced by placing the plants in a cold room at 8°C for 3 days. Water stress was imposed on detached pea leaflets, as previously described; !~4~briefly, the samples were placed on filter paper and dehydrated in air tbr several hours. This dehydration treatment was done in the dark at room temperature (around 22°C) and at a relative air humidity of around 50°~. Control leaf samples were kept on wet filter paper under the same conditions. Water potential of dehydrated leaflets, determined with a hydraulic press apparatus, i26i was around - 17 bars. Chlorophyll fluorescence measurements In vivo Chl fluorescence emission from leaves exposed to normal air with 0.03% CO~ was measured at room temperature using a twin channel modulated Chl fluorescence system (Hansatech Ltd) connected to a micro-processor power supply (Electro-Automatik, model EA-3040). This measuring system has been described elsewhere.'3i A typical example of a modulated 685-nm fluorescence curve obtained with our fluorometer, showing the different Chl fluorescence levels used in the present work, is presented in Fig. 1A. Briefly, Chl fluorescence was excited with a modulated yellow light LMoD (peak wavelength, 585 nm; modulation fi~equency, 870 Hz) provided by an array of yellow light-emitting diodes combined with an Ealing Beck 35-5404 filter. Fluorescence emission was detected with a photodiode through a 685-nm interference filter. The main advantage of modulated fluorometry is that, due

CONSTANCY OF STEADY-STATE FLUORESCENCE IN LEAVES to synchronous detection, only fluorescence induced by the pulsed light is measured, thus allowing one to monitor the influence of various non-modulated lights which do not create any measured signals. The intensity of the modulated light beam was sufficiently low (<0.025 W / m 2) so as not to induce any variable fluorescence; thus, the fluorescence level recorded was close to the basic fluorescence level (Foa). We determined that the application of a background far-red light LFR (ca 10 W / m 2) was not able to lower the fluorescence level below that observed with the measuring beam on. Short pulses (1 sec) of supersaturating blue-green light Ls (500 W / m 2 of photosynthetically active radiations) were used to measure the maximal level of modulated Chl fluorescence in dark- and light-adapted leaves (Fmd and Fn,l, respectively). It was determined that the Ls intensity was saturating (for fluorescence): application of an intense light pulse to a leaf adapted to this light intensity (500 W / m 2) did not induce any fluorescence peak (i.e. Fs = Fml and qe = 0; see also Fig. 3). Variable fluorescence was induced by a non-modulated (actinic) blue light LA, the intensity of which was adjusted from 0 to 500 W / m 2 using neutral density filters. After prolonged illumination of the leaves with LA, tile fluorescence yield reached a stable and low steady-state level Fs. The basic fluorescence yield Fol of those light-adapted leaves was determined by simultaneously turning off L A and applying a 2-sec pulse of far-red light (LFR). Calculation of the Rx, qe and qNF parameters (cfi below) requires an accurate determination of the Fol level. We assumed that the measurement of F,,1 was thst enough to avoid relaxation of the quenching processes established under light conditions, thus allowing measurements of the true dark level of the light-adapted leaf. This assumption was supported by the finding (Fig. 1B) that application of an intense light pulse Ls on the Fol level (just after LA was switched off') gave a fluorescence peak with the same height as that of the Fml level. The non-modulated lights LFR, LA and Ls were supplied by Schott KL1500 light sources combined with adequate filters (320- to 620-nm broad-band filter for L A and Ls or 730nm intert~rence filter for LFR) and were delivered via fiber optics to the leaf placed in the Hansatech leaf-clip. The fluorescence signals were displayed

25

on a potentiometric chart recorder or stored on diskettes. All light intensities were measured with a YSI-Kettering 65A radiometer; all the intensity values given in this paper correspond to light intensities at the leaf surface. The so-called photochemical (qp) and nonphotochemical (qNP) Chl fluorescence quenching coefficients were calculated according to BILaER and SCHREIBER(2): qe = (F,,,~-Fs)/(Fm~-Fo,) 1 - qNe = ((Fro, - Fol)/(Fmd - Fod)) (Fo<~lFo,). RESULTS

Figure 1A shows a curve of modulated Chl fluorescence emitted by an attached pea leaf illuminated with very weak modulated yellow light. Upon application of the actinic light LA (165 W/m2), modulated Chl fluorescence disp l a y e d the typical Kautsky effect, (19! i.e. a fast increase in the fluorescence yield followed by a slow quenching towards a low steady-state level Fs. A stable Fs level was usually reached after about 10-15 min of illumination. It can be seen that the final Fs level was very close to the fluorescence level measured in the dark before light adaptation (boa). When LA was switched off, Chl fluorescence dropped almost instantly to a lower (dark) level Fol and then rapidly rose to a stable level Foa. which, as shown in the insert of Fig. 1A, was also identical to Fs (and consequently to Foa). Thus, the Chl fluorescence traces shown in Fig. 1A seem to indicate that, independently of the fact that the leaf sample was adapted to the light or to the dark, its steady-state yield of in vivo Chl fluorescence remained constant (Fs ~ Nod ~ Nod'), The maintenance of this constant fluorescence yield was, however, accompanied by a considerable quenching of both the initial (dark) and maximal (light-saturated) fluorescence levels after light-adaptation. In other terms, F~,l and fml << Foe and Fma. Figure 2 shows that the capacity for maintaining a constant steady-state level of in vivo Chl fluorescence was not dependent on the intensity of the actinic light L a. The F s levels of the induction curves obtained with various saturating (390 W/m 2) and non-saturating (from 4 to 200 W/m 2) LA intensities were very similar, although the time

M. HAVAUX et al.

26

required to reach Fs differed from one curve to another. This effect is clearly shown in Fig. 3, a compilation of the fluorescence d a t a obtained in a large n u m b e r of m a t u r e pea leaves illuminated with different light intensities ranging ti~om 0 to ca 500 W/m2; fluorescence ratios R x = ( F x - F o d ) / ( F m d - - F o d ) (with Fx = Fs, Fro,, Fd, Fod,) were plotted against the l o g a r i t h m of the LA intensity. T h e r e are two reasons for this presentation. Firstly, the use of' the Rx ratios allowed light-induced changes in the fluorescence levels to be examined relative to the m a x i m a l possible changes in fluorescence intensity (from the d a r k level Foal) which, as a result of the nor-

malization, can take values of between 0 (-R,,d) a n d 1 (=R,,d). T h e basic and m a x i m a l fluorescence levels, b,,d a n d Fred, of all the d a r k a d a p t e d samples were a r b i t r a r i l y fixed to 0 and 1, respectively. Secondly, since the a m p l i t u d e of the m a x i m a l v a r i a b l e fluorescence (Fred- Foal)/Fod differed slightly from one leaf to another, it was found necessary to normalize the fluorescence d a t a to the (bmd -- Foal) a m p l i t u d e in o r d e r to allow comparison a m o n g samples. A n estimation of this v a r i a b i l i t y is given by the - F,,a/(Fmd - Foal) values which correspond to the 0 level. T h e low stand a r d deviation (S.D. = 0.01416) o b t a i n e d for the - Fod/(Fmd-- Fod) ratios (mean, -- 0.2341) indi-

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FIG. 1. A. Typical curve of modulated Chl fluorescence emitted in arbitrary units (a.u.) by an attached pea leaf, showing the different fluorescence parameters used in the present work. After dark-adaptation for 20 rain, the dark level (F,,d) of modulated 685nm Chl fluorescence was elicited by a weak modulated light LMOD ( ] on). Application of short pulses of intense blue light Ls ( ~ ) allowed the maximal fluorescence emission (F,,~) to be measured in those dark-adapted samples. The Kautsky effect was induced by illuminating the leaf with a non-modulated (actinic) blue-green light La (~ on; off). In the present case, the intensity of La was 165 W/m 2. After prolonged illumination with LA, Chl fluorescence reached a low steady-state level Fs. The maximal (b],,L)and dark (/~:,~)fluorescence levels in the steady state were obtained, respectively, by applying a saturating light pulse Ls or by simultaneously switching off LA and applying a pulse of far-red light Lvk ( ~ ). The /~Ld, level is the fluorescence yield obtained after readaptation of the leaf to the dark for around 30 sec, as shown in the insert. B. Effects of intense light pulses ( ~ ) on the steady-state (Fs) and dark (Fol) levels of modulated Chl fluorescence in pea leaves adapted to two different intensities of the actinic light (500 and 110 W/m2). The black arrow ~ indicates that the actinic light was switched off.

CONSTANCY OF STEADY-STATE FLUORESCENCE IN LEAVES

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cates that the heterogeneity of our plant material was rather small. A semi-log plot allowed a better examination of the Rx values at low light intensities. T h e Rs values were observed to be nearly constant over the range of intensities examined, i.e. Rs (mean value, 0.0153) was centered around Rod = 0. A statistical comparison of the N o d and Fs values by a one-way A N O V A indicated that their mean values were not significantly different at the 0.001 level: observed Snedecor variable Fou~ ( = 9.853) < F0.999 (degrees of freedom = 1, 46). T h e level (Rod,) measured after light-adapted leaves were re-adapted to darkness for a short time (ca 30 sec) was also similar to Roa and Rs, except at light intensities below about 25 W / m 2 where Rod, was intermediate between Rot and Rs. In this low light intensity range, the recovery time was observed to be substantially longer (of. below, Fig. 7). Dark-adaptation times of several minutes resulted in a complete recovery. In contrast to Rs, the "extreme" fluorescence levels (Rml and Rol) were strongly affected by light. The basic fluorescence (Rol) exhibited a roughly linear decrease with increasing light intensities whereas the light-dependence curve of Rml was biphasic, with a first linear part at low light intensities (from 0 to around 25 W / m ~) and a sharp

Fla. 3. Effect of the intensity I (in W/m 2) of the actinic light LA on the Chl fluorescence ratios Rx = (Fx - Fod)/ (F,11d- F,,d) (with the suffix X = ol, s, ml or od') measured in mature pea leaves. Note that the x-axis is logarithmic; log~ was used because intensities were in general increased by a factor of 2. Each set of experimental data (F,i, Fs, bm~, ...) corresponds to a different leaf. The -F,~/(F,n,,-P~,e) values (A) indicate the O level. ([~), Rs; (m), Rm,; (0), Ro,; (O), R,.a,. The meaning of the different symbols is also indicated on the schematic fluorescence curve shown in the insert (T and J,, actinic light on and off; t, Ls pulse)•

decrease at intensities higher than 25 W / m 2. U n d e r high light conditions (for example, 500 W/m~), Rs = R,,I, indicating that QA was fully reduced (qe = 0) and all the PS I I traps were closed. This Rs value measured in light-saturated leaves was identical to the modulated fluorescence level of a dark-adapted system having all its reaction centers open (Rod) . Thus, whether photochemistry was light-saturated or not, in vivo Chl fluorescence yield remained unchanged, clearly indicating that there is no simple, unequivocal relationship which links steady-state fluorescence emission F s and photosynthetic activity. W h e n Chl fluorescence was monitored at a wavelength

M. HAVAUX et al.

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higher than 685 nm (ca 800 nm, data not shown) where the contribution of PS I is believed to be amplified, the signals had a lower amplitude but their dependence on the actinic light intensity showed the same trends as those exhibited by 685-nm fluorescence emission• Fluorescence homeostasis was observed in a variety of plant species including spinach (Fig. 4), wisteria, lime, etc, (data not shown). T h e phenomenon was observed in plants grown in the field and in growth chambers, provided, however, that mature, well-developed leaves were used. W h e n in vivo Chl fluorescence was measured in young leaves (Fig. 4), large deviations from the ideal homeostatic behavior were observed. In very young spinach leaves for instance, a 50% increase in the Fs level relative to that of the Fod level was observed at certain light intensities. Constancy of fluorescence yield under steadystate conditions was also a characteristic of healthy leaves. It was lost as soon as the plants were exposed to stressful conditions. Figure 5 presents the effects of different environmental stress treatments, namely chilling stress (Fig. 5A) and rapid water stress (Fig. 5B), plotting relative Fs values (Fs/Fod) vs the logarithm of the actinic light intensity in pea leaves. It was observed that, in both cases, the stressed leaves were unable to adjust efficiently the steady-state fluorescence yield to the dark fluorescence Foa yield, as was the case in control leaves. This loss of homeostatic behavior occurred in different intensity ranges for

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the two types of stress. For chilled leaves, the effect was very pronounced at relatively low light intensities, between around 4 and 60 W / m 2 (Fs/Fod > 1) or at high intensities above around 300 W / m 2 (Fs/Fo~ < 1). In contrast, in desiccated pea leaflets, Fs deviated from the homeostatic level towards lower values in a larger range of light intensities (I > 20 W/m2). Observed changes in the Fs/Foa ratio originated from alteration in Fs but not in Fod yield; no significant differences in Fod were measured between young and mature leaves or between stressed and nonstressed leaves. T h e photochemical (qF) and non-photochemical (qNe) components of Chl fluorescence in control and dehydrated pea leaflets were cal-

CONSTANCY OF STEADY-STATE FLUORESCENCE IN LEAVES

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FIG. 6. Effects of the intensity of the actinic light La (I in W/m 2, logarithmic scale) on the photochemical (qe) and non-photochemical (qNe) Chl fluorescence quenching coefficients in pea leaves dehydrated for 0 (Q), O) or 2.5 hr (F1, i ) . culated according to BILGER and SCHREIBE]~i2/ from the fluorescence levels monitored in the leaves used to plot Fig. 4B. As expected, qp and qNP were markedly influenced by light intensity (Fig. 6): in low light, qp was dominant whereas, in high light, it strongly decreased (reaching the 0 value at saturating light intensity) so that q~p became the major quenching component at light intensities higher than around 65 W / m 2. It was, however, not possible to distinguish clearly between control and dehydrated leaves on the basis of this quenching analysis. In the case of leaves subjected to low temperature stress, the observed changes in steady-state modulated fluorescence (Fig. 5A) were associated with some changes in the Chl fluorescence quenching parameters (data not shown). The results presented above were obtained in leaves illuminated with different intensities of an actinic blue-green light• However, homeostasis was also observed when leaves were adapted to different light qualities. In Fig. 7, pea leaves were forced to undergo transitions from state I I towards state I and vice versa by illuminating them with a continuous far-red light LFR (absorbed predominantly in PS I) and/or with a low-intensity blue light LA (absorbed preferentially in PS II), as in Ref. 23. Sudden changes in light quality (LA to L A+ LFR, L A+ LFR to LA) resulted in marked changes in fluorescence intensity but, when the samples were allowed to adapt for a prolonged period of time (10 rain or so)

In vivo Chl fluorescence emission from intact plant leaves is a complex function of various photosynthetic processes. It is governed by the redox state of QAIgi--a phenomenon which is usually analyzed using the so-called photochemical fluorescence quenching qp/2/although there is no linear relationship between QA redox state and variable Chl fluorescence/22/ (see also Equation (4) below). Chl fluorescence can also be quenched by processes which are non-photochemical in nature. The major one is the "energy"-dependent quenching which is linked to the energization of the thylakoid membrane by the light-driven proton uptake. (5'2t) Increase in the competing thermal energy dissipation has been suggested to explain the A pH effect, ia'2°i although this hypothesis awaits experimental support. (~3) Other known non-photochemical quenching mechanisms include the state transition-related quenching, resulting from a change in the amount of absorbed light energy channelled to PS II, (~°'23/and the decline in fluorescence yield associated with photoinhibition of PS II under high-light conditions. (7; The extent of those different quenching processes and their relative proportions are strongly influenced by light intensity/~Si (see also Fig. 6). Despite this complexity, Chl fluorescence is usually maintained at a relatively low level in light-adapted leaves, i2'~~,~9iOur data demonstrate that the steady-state level of in vivo Chl fluorescence excited by a yellow modulated light is constant and virtually independent of the intensity/quality of the actinic light used to drive photosynthesis. Examination of Figs 4 and 5 indicates that, in well-developed leaves, the maximal measured deviation of the Fs level from the dark level Fod was largely below 10% . This maintenance of a near constant, light intensity-independent F s level by light-adapted leaves provides an interesting example of biological homeostasis• From a thermodynamic point of view, the Fs

30

M. HAVAUX et al. LfLar

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FIG. 7. Effects of an additional non-modulated far-red light LFR (10 W/m 2) on the steady-state level of modulated Chl fluorescence in a pea leaf illuminated with a low-intensity continuous blue light L A (10 W/m2). The dotted line indicates the basic fluorescence level Fod of the dark-adapted leaf. Fs was the steady-state Chl fluorescence obtained after 20-rain illumination of the leaf sample with L A. fluorescence level represents the attractor of the system, i.e. the level to which the system naturally settles. (u T h e attractor is the thermodynamic optimal state where the state-change force is zero and the biological system is in full " h a r m o n y " with its environment. T h e state-change associated with the evolution of the system towards the attractor level is reflected by the drastic modification of the R,,I~ and Ro~ fluorescence ratios, which mark the limits of variation of Rs in a given state. For a simplified model of PS lI, considered as an assemblage of a reaction center (denoted by the subscript b) and a pool of Chls (subscript 2), it has been demonstrated (~6i that the theoretical expressions of F<,I, Fmi and Fs are: Vo~ = J ~ 2 v / ( 1 - G)

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(4)

where J2 is the light absorption flux in PS II, P2r is the probability of fluorescence, G is the probability of energy exchanges between PS II units (=P22), T is the probability of energy cycling between the reaction center and the Chl pool (=P2bPb2), V = 1 - qe is the relative variable fluorescence and B is the fraction of closed PS I I reaction centers. Equation (3) shows that Fs is a function of (a) the energy input of PS II (.]2), (b) the conformation of the system (i.e. the rate constants of all the energy transfer reactions) reflected by the probabilities P2i, and (c) the a p p a r e n t behavior of the system (i.e. B which is determined by the balance between the excitation of the PS I I traps and the reactions downstream involved in the reopening of the closed centers). T h e strong light dependence of the Fml and F<,~ levels (Fig. 3) indicates that steady states induced by different actinic light intensities were characterized by a different conformation [see Equations (1) and (2)]. Thus, it is very intriguing that, under any steady-state conditions, the values of the PS II rate constants were adjusted in such a way that the complex expression given in Equation (3) remained constant.

CONSTANCY OF STEADY-STATE FLUORESCENCE IN LEAVES Whether fluorescence homeostasis has a precise physiological function remains an open question. It is hard to understand the profit plants can reap from maintenance of the same fluorescence yield at all light intensities. One would logically expect a lower yield at light intensities limiting for photosynthesis in order to optimize the efficiency of light utilization, and a higher yield at light intensities close to the saturation level in order to reduce the risk of photoinhibition. Tile role of fluorescence (which represents not more than 5% of the absorbed light energy) as a valve dissipating excess energy seems to be negligible in vivo. This latter function is possibly devoted primarily to non-radiative energy dissipation---a hypothesis supported by the finding that exposure of pea leaves to photoinhibitory light was accompanied by a drastic increase in photoacoustically monitored heat emission./6'12) The molecular mechanisms responsible for adjustments of" in vivo Chl fluorescence emission are beyond the scope of this paper. However, the fast reversibility (within seconds) of the FoI level (Fig. 1A and B) and the /~ml level (not shown) would argue for a prominent role of the fast relaxing transthylakoid proton gradient A pH, at least at moderate and high light intensities. At low light intensities ( < ca 25 W/m2), complete recovery was still observed but needed noticeably longer times (in the range of several minutes), suggesting that a much slower phenomenon, such as the protein (de)phosphorylation associated with the state I state I I transitions, took precedence over A pH. Support for the involvement of the state transition phenomenon in the homeostatic regulation at low light irradiances is clearly demonstrated in Fig. 7 where leaves were adapted to different weak lights which, due to their spectral characteristics, induced phosphorylation (LA) or dephosphorylation (LA + LFR) Of"the light harvesting Chl protein complexes (cf. Ref. 23). An obvious consequence of fluorescence homeostasis is that usefhl information concerning the efficiency of photon utilization in photosynthesis cannot be directly derived from in vivo measurements of steady-state Chl fluorescence emission, since this latter signal was constant whether the photosynthetic reaction centers were open and capable of engaging in photochemical work or closed by light saturation.

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When leaves were exposed to unfavorable environmental conditions (Fig. 5), the homeostatic behavior was lost. Comparison of Figs 5B and 6 (control vs stressed leaves) shows that different steady-state fluorescence intensities were associated with similar qv and qNP values, suggesting that fluorescence homeostasis was more sensitive to perturbations of the physiological state of leaves than the familiar quenching coefficients. The in vivo measurements of the "bioenergetic homeostasis" phenomenon presented here could be a very useful approach for the early detection of subtle changes in the state of health of plants. The differential response to environmental stresses of F s and the fluorescence quenching parameters also suggest that qp and qNP might not be the ideal representation of the various factors which modulate the steady-state yield of in vivo Chl fluorescence level in intact leaves. A theoretical analysis of the physical meaning of those fluorescence parameters, which is presented in a separate paper,/16i partially confirmed this idea. REFERENCES

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