Photosynthesis Research 27: 135-142, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands. Regular paper

Chlorophyll fluorescence and photoinhibition in a tropical rainforest understory plant Jean-Luc Le Gouallec 1, Gabriel Comic 2 & Jean-Marie Briantais Laboratoire d'Ecologie V~g~tale Bftt. 362, Universit~ Paris 2(1, 91405 Orsay, Cedex, France; 1Permanent address: Laboratoire de Botanique Tropicale, UniversitO Paris VI, 12 rue Cuvier, 75005 Paris, France; 2To whom correspondance has to be sent Received 2 August 1990; accepted in revised form 19 November 1990

Key words:

chlorophyll fluorescence, Elatostema repens, photoinhibition, photosystem II, recovery

Abstract

The data presented here deal with the effects of high-light exposure on the 77 K fluorescence characteristics of Elatostema repens. It is shown that the decrease of the variable fluorescence during the treatment is biphasic. The reactions responsible for the first phase of fluorescence quenching are saturated under 700/zmol photon m -z s -I and insensitive to streptomycin, whereas those responsible for the second phase are not yet saturated under 700/~mol photon m -2 s -1 and sensitive to streptomycin. It is concluded that only the second phase of fluorescence quenching is associated with photoinhibitory processes. Rate and amplitude of recovery from photoinhibition are maximum under very low light (3.5 ~mol photon m -2 s-~), and very small at a moderate light (160/xmol photon m -2 s -1) which does not cause photoinhibition. It is concluded that recovery processes are inhibited during photoinhibition. It is suggested that they could be associated with damage occuring on the oxidizing side of PS II.

Abbreviations: Fo, Fv, F m - initial, variable and maximum fluorescence, respectively; PFD - photon flux density; PS II - photosystem II

Introduction

Exposure of photosynthetic organisms to excess of light results in photoinhibition of photosynthesis. This photoinhibition occurs mainly as a consequence of damages at the PS II reaction centers (Bjrrkman 1987). It is believed that photoinhibition results from an imbalance between continuous degradation and repair of some PS II proteins. Under high light the rate of degradation is in excess of that of repair resulting in decrease in PS II activity (Greer et al. 1986, Samuelson et al. 1989). Non-radiative (thermal) dissipation of excess energy during high light can protect primary photochemistry from photoinhi-

bition. This dissipation of excess excitation energy may involve the conversion of the xanthophyll pigment, violaxanthin to zeaxanthin (Demmig et al. 1987). The study of changes in low-temperature (77 K) chlorophyll fluorescence is now widely recognized as a unambiguous way to assess damage caused by excessive light (photoinhibition). Indeed, the linear relationship between lowtemperature chlorophyll fluorescence and quantum yield of photosynthesis is well established, enabling fluorescence to be used as a quantitative measurement of photoinhibition of PS II photochemistry (Bj/Srkman 1987, Demmig and Bjrrkman 1987).

136 Most of the studies concerning photoinhibition have been made on sun plants grown in shade. Few reports have been concerned with photoinhibition occuring in shade plants. Elatostema repens is an understory plant native to South East Asia. This shade plant has been previously anatomically studied (Le Gouallec et al. 1986). The effect of high-light treatment on gas exchange and PS II electron transfer activity have also been examined (Le Gouallec and Cornic 1988). It was then observed in cells isolated from high-light pretreated leaves a significant decrease of water to phenylbenzoquinone electron flow rate, measured in both limiting and saturating light intensity only after the first hour of pretreatment. After this lag, extents of inhibition were the same in both limiting and saturating light. It was also observed that in the presence of streptomycin during the high-light pretreatment, the initial lag of PS II activity inhibition was cancelled. From these data it was proposed that photoinhibition results in a decrease in number of active PS II centers after a lag during which repair or new synthesis from a pool of m-RNA compensate degradation. In the present work the changes in chlorophyll fluorescence induced by a photoinhibitory high-light (700/xmol m -2 s -1) treatment and during a subsequent recovery period are analysed. The importance of protein synthesis is also examined.

Materials and methods

Plant material Cuttings of Elatostema repens Lour. (Hall). f. were obtained from plants grown in a glasshouse under shaded conditions. Rooted cuttings were transferred, at least one month prior to experiments, to a growth chamber where the climatic conditions were as follows: photoperiod 16 h and constant temperature of 24°C. Inside the growth chamber, plants were kept in a dark plexiglass box. Neutral filters were placed on the open-top of the box to give a Photon Flux Density (PFD) at the plant level of 40/~mol m -2 s -1 as measured with a quantum sensor (LI-COR, Lincoln, NE, USA). Air humidity was approaching saturation. The light was supplied by metal halide lamps (OSRAM HQI T400W/DH). Plants were

grown on vermiculite and watered daily with deionised water and with Hoagland type nutrient solution. Only the five fully expanded terminal leaves of a growing apex were used for experiments.

Fluorescence measurements The fluorescence measurement system was similar to that described by Powles and Bj6rkman (1982) with a light source provided by a slide projector lamp (24 V OSRAM) powered by a regulated DC power supply. The excitation light was filtered at 470 nm. Fluorescence measurements were performed in liquid nitrogen on leaf discs. PS II fluorescence emission was measured at 690nm. A five minute dark adaptation in normal air at room temperature was given before the addition of liquid nitrogen. The measurement was performed 2 min after this addition. Photoinhibitory treatment and recovery All the photoinhibitory treatments reported here were given in normal air at 24°C. The photoinhibitory treatment, consisting of exposures to 700/xmol m - 2 s -1, was given in the growth chamber either on attached leaves or on discs floating on deionised water. Photoinhibitory treatments were also given on leaf discs placed in the compartment of the Hansatech leaf discs 0 2 electrode. Similar results were obtained by these various methods. In some experiments the 700/.~mol m - 2 s -1 photoinhibitory treatment was given on leaf discs in the growth chamber in the presence of the protein translation inhibitor, streptomycin (4g1-1) and tween (1%). A photoinhibitory treatment at 1400 p~mol m -2 s -1 was also carried out using floating discs placed inside a temperature-controlled assimilation chamber.

Results

Effects of photoinhibitory treatments on fluorescence characteristics The time courses of the changes in F m and F o upon 700/xmol m - 2 s - 1 illumination of leaves (Fig. 1), demonstrates that F m is strongly decreased by high light, whereas F o is not signifi-

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Fig. 1. Kinetics of fluorescence parameters during an exposure to 700/xmol m 2 s-~. O, F m after treatment given on leaf discs floating on deionised water; &, F m after treatment given on attached leaves; ©, F o after treatment given on leaf discs floating on deionised water; Lx, F, after treatment given on attached leaves; [] Fv/F ~, for attached leaves, • for leaf discs.

cantly affected. From a semi-log plot of these fluorescence changes, presented in Fig. 2, it can be seen that decreases on both F v and Fv/F m are biphasic. The initial phase seen during the first 30 min of high light is faster than the second phase. The decrease in FJFm measured in isolated cells shows the same biphasic kinetic as that observed in leaf discs. Streptomycin had no sideeffects on photosynthetic capacity as evidenced by photosynthetic 0 2 evolution measured in a leaf discs 0 2 electrode (data not shown). Figure 3 shows the kinetics of the F v / F m decrease plotted as the log of the percent of the initial value, for a 1400/xmol m - 2 s -1 and 700/~mol m -2 s -1 exposures of different durations in the presence or absence of streptomycin. As Fig. 1, these treatments induced a F m decrease, while F o remained unchanged (data not shown). It is evident from Fig. 3 that irrespective of the light treatment, the decrease is composed of two phases and that the first phase is the same in all cases. It is only after about 30 min that differences appear between treatments. This means that the first phase is not the sum of two

kinetics, i.e., there are two distinct phenomena: one common decrease ending after 30min of high-light exposure, the other starting after the end of the first decrease. I

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TIME (h) OF EXPOSURE Fig. 3. Kinetics of Fv/Fm expressed as the log of percent of the initial value during an exposure to: O, 700/xmol 700/~mol m -2 s -1 in the presence of streptomycin; II, 1400/~mol m -2 s -~. Linear regression equations: 1400/zmol m -2 s-l: 700/zmol m -2 S -1" y = -0.00195 (x, rain) + 1.999 (phase 1) y = -0.00194 (x, min) + 2.002 (phase 1) y = -0.00160 (x, min)+ 2.003 (phase 2) y = -0.0024 (x, min) + 1.945 (phase 2) First phase, all results: 700 ~mol m -2 s -1 in the presence of streptomycin: y = -0.00182 (x, min) + 1.999. y = -0.00164 (x, min) + 1.997 (phase 1) y = -0.00093 (x, min) + 1.985 (phase 2)

Increasing the PFD of the photoinhibitory treatment from 700 to 1400 ~mol m -2 S - 1 c a u s e s a larger decrease of the Fv/Fr~ ratio. Thus it appears that the level of damage is dependent of the PFD prevailing during treatment. This is what one would expect in the case of photoinhibition; a light-dependent damage of the photosynthetic apparatus. If the 700/xmol m -2 s -1 exposure is given in 'the presence of streptomycin, the decrease of the Fv/F m ratio is faster in the second phase. Thus inhibition of protein synthesis enhances the extent of photoinhibition as measured by the decrease of the F v / F m ratio. This has already been observed in Phaseolus vulgaris (Greer et al. 1986) and with PS II electron transfer activity on Elatostema (Le Gouallec and Cornic 1988) and on Anacystis nidulans (Samuelson et al. 1987). Recovery of Fv/Fm after 3 h exposure to 700 txmol m -2 s -1

The recovery kinetics of Fv/F m after 3 h exposure

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Fig. 5. K i n e t i c s of F v / F m r e c o v e r y a f t e r 3 h u n d e r 7 0 0 / x m o l m -2 s ~ as a f u n c t i o n of the d u r a t i o n of the d a r k p e r i o d in a s e q u e n c e (3.5 ~ m o l m -z s -1 d a r k ) d u r i n g r e c o v e r y . T h e d u r a t i o n of the 3.5 ~ m o l m -2 s ~ p e r i o d b e f o r e d a r k is i n d i c a t e d in b r a c k e t s in min.

1986), Sinapsis alba (Borge et al. 1987), and Chlamydomonas (Ohad et al. 1984). The recovery is maximal when the PFD prevailing during the recovery phase is as low as 3.5 Izmol m -2 s -1. It must be noted that even in this case the recovery is only partial since the initial F v / F m value was 0.80 and the level of maximum recovery is 0.71 (about the level corresponding to the start of the second decrease phase). Figure 5 shows that with a low light (3.5/zmol m-Zs-1)-dark sequence during the recovery phase the level of recovery is controlled by the duration of the low light period.

Discussion and conclusion

Two phases in fluorescence quenching during high-light treatment It is evident from the results shown (Figs. 1 and 2) that the decrease of the F v / F m ratio is due solely to a decrease of F m , since F o remains unchanged during the treatment. This quenching is clearly biphasic, contrary to the quasi-first order kinetics obtained by Demmig and Bj6rk-

man (1987) on other plants. It should be noted that such a monoexponential decay also occurs during illumination at 1400 ~mol m -2 s -1 (Fig. 3). Thus, in Elatostema repens, there are two phases of fluorescence quenching when leaves are submitted to high-light treatment. The two phases differ in respect to their response to light and to streptomycin. As shown by Fig. 3, the reactions responsible for the first phase are saturated under a PFD 700 izmol m - 2 s -1. Incr_easi_nlg the PFD from 700 to 1400 ~mol m s dramatically increased the rate of fluorescence decay during the second phase but did not change that during the first phase. Figure 3 also clearly indicates that with a photoinhibitory treatment at 700/zmol m - 2 s - 1 the first phase of fluorescence quenching is insensitive to streptomycin whereas the second is streptomycin sensitive. Horton and Hague (1988) (see also Genty et al. 1990) showed that 2 'irreversible' quenchings are photoinduced, which saturations are reached at very different light intensities. First phase of F m decrease may correspond to the quenching which is saturated at lower intensity and could not be associated to destruction of PS: centers.

140 A comparison of the results in Fig. 2 to data previously published (Le Gouallec and Cornic 1988) shows that the first phase of fluorescence quenching is not correlated with a decrease of PS II photochemical activity measured either at limiting or saturating light. During the first hour of high-light treatment the decrease in F m which is not accompanied by inhibition of PS II photochemistry is a situation very similar to the one observed (Etienne and Lavergne 1972) upon addition of low concentration of dinitrobenzene. According to Butler's model (Butler and Kitajima 1975) only an increase of kd (i.e., of thermal deactivation at the level of excited P+680) will occur during this first hour. It could be also suggested that the maintenance of PS II activity during the first hour of high light would be the result of protein synthesis from an existing pool of m-RNA which would then be slowly depleted. Inhibiting translation under high light by streptomycin addition would bring about an immediate decrease of PS II activity. The second phase of fluorescence quenching which starts after 30 min to 1 h high light, presents 3 features which are in agreement with what is thought to be a PS II photoinhibition (Powles and Bj6rkman 1982, Demmig and Bj6rkman 1987). Firstly, it is related to a decrease of PS II activity (Le Gouallec and Cornic 1988). Secondly, it is dependent on PFD not being saturated at 700/zmol m -2 s -1 (Fig. 3). Thirdly, it is dependent on protein(s) synthesis. This second phase of F m quenching corresponds to a decrease of active PSII centers, indeed both activities in limiting and saturating light intensities are equally inhibited. As no change in F o was seen, fluorescence of these inactivated centers must be blocked in F o. Thus they are characterized by a non-radiative deactivation which yield is equal or close to the yield of PS II photochemistry. Such a state of PS II is typical of center which electron donor side is blocked. Then we would like to suggest as Jegersch61d et al. (1990) that photoinhibition in vivo results from an inhibition of donor side of PS II. This suggestion will be reinforced by results reported here on the light-dependent recovery after photoinhibition which are discussed below.

Recovery of fluorescence yield after photoinhibition As shown in Fig. 4, the Fv/F m recovery high-light treatment (1) requires light as already noted by others (Greer et al. 1986) and (2) is strongly dependent on the PFD prevailing during the recovery period. A very low PFD (about half of that necessary to reach the light compensation point of leaf net CO 2 uptake in normal air) is required for maximal recovery. This illustrates the very low energetic requirement of the process in this plant. In contrast, the recovery is very small or absent under a PFD of 160/zmol m -2 s -1 which is not photoinhibitory in this plant and is closed to the light saturation point of photosynthesis (Le Gouallec 1988). Continuous light is required for the recovery (Fig. 5) of the F v / F m ratio since the degree of recovery after the photoinhibitory treatment is dependent on the duration of exposure to light (3.5/zmol m -2 s-l). It should also be noted that when the recovery of PS II electron transport activity is complete (Le Gouallec and Cornic 1988), the Fv/F m ratio has not fully recovered. Nevertheless, the recovery kinetics of the F J F m ratio and the PS II electron transport activity (Le Gouallec and Cornic 1988) are similar under the growth irradiance (40/zmol m -2 s-~). Thus, the reversal of the quenching is likely to be related to PS II restoration. After 2 h of recovery, the value of F v / F m is higher in the dark than under 160/zmol m -2 s -1. Thus, clearly, repair process(es) is (are) already inhibited by moderate irradiance. Thus, it appears that the decrease in the F v / F m ratio in this plant upon exposure to high light is both due to photoinhibitory damage and inhibition of repair process(es). This conclusion is very similar to that of Greer and Laing (1988) who studied photoinhibition and recovery on kiwifruit plants. This conclusion raises the question of the effect of streptomycin during high-light treatment (Fig. 3). The increase in the susceptibility of the photosynthetic apparatus to high light in the presence of streptomycin can be easily interpreted using the notion of a balance between repair and destruction processes during photoinhibition as proposed by Kyle et al. (1984) and

141 u s e d a f t e r b y o t h e r s ( e . g . , S a m u e l s o n et al. 1989). H o w e v e r , as s u g g e s t e d b y t h e results of Fig. 4, t h e r e p a i r p r o c e s s e s a r e m o s t l i k e l y to b e i n h i b i t e d in high light a n d , thus, c a n n o t b e inhibited further. It c o u l d b e t h a t in t h e p r e s e n c e o f s t r e p t o m y c i n , which d o e s n o t i n h i b i t t h e s y n t h e s i s o f a specific p r o t e i n b u t r a t h e r t h a t o f t h e w h o l e cell, a n o t h e r p r o c e s s o f p h o t o i n h i b i t i o n (which d o e s n o t o c c u r u n d e r n a t u r a l c o n d i t i o n s ) is t r i g g e r e d . It c o u l d b e also t h a t d a m a g e o c c u r e d o n t h e o x i d i z i n g side of PS I I a n d t h a t it is t h e r e c o v e r y f r o m this t h a t is t h e limiting s t e p o f t h e w h o l e p r o c e s s . A s s h o w n by C a l l a h a n et al. (1986) a n d b y Jegersch61d et al. (1990) i n a c t i v a t i o n o f w a t e r - s p l i t t i n g e n z y m e c o m p l e x in l e a v e s o r isol a t e d c h l o r o p l a s t s results in an i n c r e a s e in t h e s u s c e p t i b i l i t y to p h o t o i n h i b i t i o n . This c o u l d exp l a i n w h y a m o d e r a t e light i n t e n s i t y is u n a b l e to induce photoinhibition, but blocks the recovery. This is in a g r e e m e n t with t h e s u g g e s t i o n of Clel a n d a n d M e l i s (1987) t h a t d u r i n g p h o t o i n h i b i tion t h e limiting s t e p m a y b e t h e d a m a g e a n d lysis of D 1 (which will r e q u i r e s t r o n g light a n d b e d e p e n d e n t on s t r e p t o m y c i n ) w h i c h will i n d u c e a disconnection or a destruction of the oxidizing side ( Z ) o f PS II. T h i s is n o t s u r p r i s i n g since Z has b e e n i d e n t i f i e d as t y r o s i n e 161 o f D 1 p r o t e i n ( D e b u s et al. 1988).

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Characterization of weak light photoinhibition of PS II and its light induced recovery. Plant Physiol 82:261-269 Cleland RE and Melis A (1987) Probing the events of photoinhibition by altering electron-transport activity and light-harvesting capacity in chloroplast thylakoids. Plant Cell Environ 10:747-752 Debus RJ, Barry BA, Sithole I, Babcock GT and Mclntosh L (1988) Directed mutagenesis indicates that the donor to W680 in photosystem II is tyrosine 161 of the D 1 polypeptide. Biochemistry 27:9071-9074 Demmig B and Bj6rkman O (1987) Comparison of the effect of excessive light on chlorophyll fluorescence (77 K) and photon yield of 02 evolution in leaves of higher plants. Planta 71:171-184 Demmig B, Winter K, Kriiger A and Czygan FC (1987) Photoinhibition and zeaxanthin formation in intact leaves. A possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol 84:218-224 Etienne AL and Lavergne J (1972) Action du m-Dinitrobenzbne sur la phase thermique d'induction de fluorescence en photosynth~se. Biochem Biophys Acta 283:268-278 Genty B, Wonders J and Baker NR (1990) Non-photochemical quenching of Fo in leaves is emission wavelength dependent: consequences for quenching analysis and its interpretation. Photosynth Res 26:133-139 Greer DH and Laing WA (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: effect of light during growth on photoinhibition and recovery. Planta 175:355-363 Greet DH, Berry JA and Bj6rkman O (1986) Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature, and requirement for chloroplast-protein synthesis during recovery. Planta 168:253-260 Horton P and Hague A (1988) Studies on the induction of chlorophyll fluorescence in isolated barley protoplasts. IV. Resolution of non-photochemical quenching. Biochim Biophys Acta 932:107-115 Jegersch61d C, Virgin I and Styring S (1990) Light-dependent degradation of the D 1 protein in photosystem II is accelerated after inhibition of the water splitting reaction. Biochemistry 29:6179-6186 Krause GH, Leasch H and Weis E (1988) Regulation of thermal dissipation of absorbed light energy in chloroplasts indicated by energy-dependent fluorescence quenching. Plant Physiol Biochem 26:445-452 Kyle DJ, Ohad I and Arntzen CJ (1984) Membrane protein damage and repair. Selective loss of a quinone-protein function in chloroplast membranes. Proc Natl Acad Sci USA 81:4070-4074 Le Gouallec (1989) Effet de forts 6clairements sur la photosynth6se de Elatostema repens (Urticac4es). Th6se de doctorat de l'Universit6 Paris VI Le Gouallec JL and Cornic G (1988) Photoinhibition of photosynthesis in Elatostema repens. Plant Physiol Biochem 26:705-712 Le Gouallec JL, Leclerc JC and Coute A (1986) Convergence morphologique entre cellules assimilatrices de plantes sup6rieures et d'algues vertes unicellulaires. CR Acad Sci Paris 303:677-682 Ohad I, Kyle DJ and Arntzen CJ (1984) Membrane protein

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(1987) The susceptibility of photosynthesis to photoinhibition and the capacity to recovery in high and low light grown cyanobacteria, Anacystis nidulans. Plant Physiol 83: 438-441 Skogen D, Chaturvedi R, Weidemann F and Nilsen S (1986) Photoinhibition of photosynthesis: effect of light quality and quantity on recovery from photoinhibition in Lernna gibba. J Plant Physiol 126:195-205