Planta

Planta (1987) 171:171-184

9 Springer-Verlag 1987

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of Oz evolution in leaves of higher plants* Barbara Demmig** and Olle Bj6rkman Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA

Abstract. High-light treatments (1 750-2000 gmol photons m - 2 " s -1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (FM, 692 and Fu, 734). The response of instantaneous fluorescence at 692 nm (Fo, 692) was complex. In leaves of some species Fo, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of Fo, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (Fv/FM,692) and the photon yield of 02 evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO2-free gas stream containing 2% 02 and 98% Nz under weak light (100 g m o l . m - 2 - s -1) resulted in a general and fully reversible quenching of 77 K fluorescence at 692 and 734 nm. In this case both Fo, 692 and FM, 692 were invariably quenched, indicating that the quenching was caused by an increased nonradiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in/7o, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both Fo, 692 and * C.I.W.-D.P.B. Publication No. 925 ** Present address: Lehrstuhl ftir Botanik II der Universit/it,

Mittlerer Dallenbergweg 64, D-8700 Wfirzburg, FRG Abbreviations and symbols." kDa=kilodalton; LHC-II=lightharvesting chlorophyll-protein complex; P F D = p h o t o n flux density (photon fluence rate); PSI, PSII=photosystem I, II; Fo, FM, Fv=instantaneous, maximum, variable fluorescence emission; c~=absorptance; ~b,=photon yield of 02 evolution (absorbed light)

FM, 692 This general quenching had a much longer relaxation time than reported for ApH-dependent quenching in algae and chloroplasts. Sun leaves, whose Fv/Fu, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% 02, 0% CO2). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition. Key words: Chlorophyll fluorescence (77 K) - Light (excessive) Photoinhibition of photosynthesis Photosynthesis (photon yield) - Quantum yield.

Introduction It is well known that exposure of leaves to considerably brighter light than they have experienced during their development results in photoinhibition of photosynthesis. This photoinhibition is manifested by a reduced photon yield (quantum yield) and light-saturated capacity of photosynthesis in intact leaves, a reduced electron-transport activity, and altered chlorophyll-fluorescence characteristics. There is general agreement that photoinhibition is caused by damage to the photochemistry of photosystem II (PSII) (for a review, see Powles 1984). Chlorophyll-fluorescence characteristics can provide valuable information on stress effects on

172

B. Demmig and O. Bj6rkman: Response of fluorescence and photon yield to excessive light

leaves and in recent years fluorescence techniques have been widely applied in studies on photoinhibition (e.g. Critchley and Smillie 1981; Fork et al. 1981; Powles and Bj6rkman 1981, 1982; Bj6rkman and Powles 1984; Ludlow and Bj6rkman 1984; Ogren and Oquist 1984a, b, c; Greer et al. 1986). Exposure of leaves to excessive light is known to result in a sustained reduction of PSII fluorescence emission at 77K, especially of the variable emission, Fv, which is the difference between the maxim u m fluorescence, FM, and the instantaneous fluorescence Fo. However, fluorescence quenching takes place not only when leaves are exposed to photon flux densities (PFD) in excess of those experienced during their development. Indeed, fluorescence quenching can also be observed when leaves developed in full sun and kept at a lower P F D for about 1 h are exposed to a higher PFD. Such quenching can be detected within a few seconds after the increase in PFD. This rapid reduction in Fv a n d FM, commonly termed "energy-dependent" or " A p H - d e p e n d e n t " quenching, is generally thought to be associated with a build-up of a proton gradient across the thylakoid membrane and is considered to reflect an increased rate of non-radiative deexcitation (e.g. Krause et al. 1982, 1983; Schreiber et al. 1986). A rapid relaxation of the quenching occurs upon return to a lower P F D or when the leaf is darkened, and a large part of the quenching is usually relieved within I min. However, in many leaves a component of the quenching exhibits a much longer relaxation time. For example, Bj6rkman and Powles (1984) observed that sun leaves of unstressed Nerium oleander plants, growing in the field exhibited a quenching of 7 7 K fluorescence during the day which closely followed the P F D of the sunlight incident on the leaves. Although this quenching was fully reversible upon lowering the P F D the relaxation time was in the order of 1 h. It thus appears that the reduction in variable fluorescence induced by exposure of leaves to excessive P F D s does not exclusively or necessarily reflect a detrimental effect on PSII but may result from a regulatory process that serves to dissipate excess excitation energy. The objectives of the present study were 1) to distinguish those changes in fluorescence characteristics that are related to detrimental effects of excessive light on PSII from those that may reflect the operation of regulatory process; 2) to establish a quantiative relationship between the effect of photoinhibition on 77K fluorescence characteristics and on the photon yield of oxygen evolution; and 3) to examine possible factors underlying the

difference in susceptibility to photoinhibition between sun and shade leaves. A recent study (Bj6rkman and Demmig 1987) provides evidence that the photon yield of oxygen evolution shows little variation in non-stressed leaves among vascular plants of widely diverse taxonomic groups, life forms and habitats and that the same is true for one fluorescence characteristic, namely the ratio of variable to maximum fluorescence at 692 nm (l~v/F~, 692). Hence, both the photon yield and the Fv/FM, 692 ratio could serve as excellent quantitative indicators of stress effects. In the present paper we further examine the effect of intense light on these two parameters and the relationship between them. We present further evidence for the occurrence of a fluorescence quenching that has only a small effect on the Fv/FM, 692 ratio and may reflect a regulatory process which operates on a time scale of several minutes to hours. Rate of recovery from photoinhibition in shade and sun leaves is also considered. Materials and methods Plant materials. Hedera canariens& Willd. (a green non-variegated form), Monstera delieiosa Liebm., Nerium oleander L. and Sehefflera actinophylla (Endl.) Harms were from local nursery stock. Glycine max (L.) Merrill (soybean) cv. Comsoy was grown from seed (Dekalb-Pfizer Genetics, Beaman, IO., USA). Gossypium hirsutum L. (Upland cotton) cv. Dunn was grown from seeds kindly provided by Dr. J.R. Ehleringer, Department of Botany, University of Utah, Salt Lake City, U., USA. Phaseolus vulgaris L. (bean) cv. Bush Blue Lake 274 was grown from seed supplied by Northrup King Seeds, Fresno, Cal., USA. Rhizophora stylosa Griff. (a mangrove), was grown from vegetative propagules collected in the wild at Cape Ferguson, North Queensland, Australia, by Dr. Barry Clough, Australian Institute of Marine Science, Townsville, Queensland. Typha latifolia L. was collected in a local marsh at Stanford, Cal., USA. Hedera, Monstera, Nerium and Typha were grown outdoors in soil with ample water supply. Hedera was grown both in deep shade on the north side of a building (photon flux density, PFD = p h o t o n fluenee rate=< 30 gmol.m Z.s- 1) and in full daylight ( P F D = u p to 2 0 0 0 p m o l - m - 2 " s 1). Monstera was grown in deep shade between two buildings ( P F D = 1 0 30 gmol. m 2. s - 1) while Nerium and Typha were grown in full daylight (PFD .

.o_ _ a o - o

....... o -o

Typho ---~

Monstero

0.2

0

~

Hedera

0

1,0~[

60

100 /~mol m-2s-1

08 I-o%o o~,.O__o_____L0oo =__.... ?_..,..o-O . . . . _-o

-60 0

ing exposure to a high P F D in 2% O2, 0% CO2 for a sun leaf of Typha latifolia and a shade leaf of Monstera deliciosa. The low-light period in air and the high-light treatment in 2% 02, 0% CO2 were 100 and 2000 g m o l - m 2-s 1 for Typha and 50 and 1850 p m o l . m -2 .s -1 for Monstera

120 180 240 300 360 420

2000 ffmol m-2s-1

C

0

~

60 120 180 T i m e , rain Fig. 8. Time courses of the change in the Fv/FM, 692 ratio dur-

Y

0.2

0 J~

o.g -~---'-o

O.4-

-60

0.2 I

"~

I

0.8

-60

"~'.O..

I

i,i

A I

0

[

Gossypium

60 120 180 240 500 360 420 4-80 540 Time, min

Fig. 7A-C. Time courses of the changes in the Fv/FM, 692 ratio of 77K chlorophyll fluorescence induced by exposure to high PFD in A a sun leaf of Typha latifolia and a shade leaf of Monstera deliciosa; B in sun and shade leaves of Hedera eanariensis and C leaves of Gossypium hirsutum developed at 150 and 1000 g m o l . m Z.s 1. The high-light treatments and the low-light period preceding and following the high-light treatments were 2000 and 100 g m o l . m 2-s 1 for Typha and 1775 and 28 g m o l - m - 2-s-1 for Monstera

in these comparisons since they exhibit vastly different capacities for light-saturated photosynthesis. The light-saturated rates of CO2 uptake in normal air at 25 ~ C, determined on leaves, grown under the same conditions as those used in the photoinhibition experiments, were approx. 38 and 3.5 gmol C O 2 . m - 2 - s -~ for Typha sun leaves and Monstera shade leaves, respectively. As shown in Fig. 8, a sun leaf of T. latifolia showed a markedly greater decline of the Fv/FM

ratio when exposed to bright light in an atmosphere of 2% 0 2 , 0 % C O 2 than in normal air (compare Fig. 7A), indicating that prevention of photosynthesis did indeed increase the susceptibility to photoinhibition. The difference between the two treatments was much smaller in M. deliciosa shade leaves, which is consistent with the much lower light-saturated photosynthetic capacity of these leaves. However, a clear difference in the response to high P F D still remains between the Typha and the Monstera leaves, even when photosynthesis was prevented (Fig. 8). In Typha the FvJ FM, 692 ratio tended to reach a plateau after 2-3 h in bright light, whereas in Monstera this ratio continued to fall with quasi-first-order kinetics. A summary of the effect of a 3-h treatment at a high P F D and 2% 02, 0% CO2 on different species grown in direct daylight and in the shade is given in Table 3, It is evident from the much greater decline in the Fv/FM, 692 ratio that the shade leaves were more susceptible to photoinhibition than the sun leaves even when photosynthesis was largely prevented. Hence, in addition to any protective effect of a greater dissipation via photosynthesis, sun leaves may possess other properties that make them less susceptible to photoinhibition than shade leaves.

Comparison of rates of recovery in sun and shade leaves. The recovery of the Fv/FM, 692 ratio that takes place after photoinhibitory treatments of a

180

B. Demmig and O. Bj6rkman: Response of fluorescence and photon yield to excessive light

Table 3. Chlorophyll-fluorescence characteristics (77K) at 692 nm before and after a 3-h exposure to a high PFD (1900 gmol .m -2. s-t) in 2% 02, 0% COz for different species and growth light regimes Species

Growth light regime

Monstera delieiosa

Deep shade

Phaseolus vulgaris

300 gmol .m - 2. s 1

Glyeine max

Winter daylight

Typha latifolia

Summer daylight

Nerium oleander

Summer daylight

Hedera canariensis

Summer daylight

Before After Before After Before After Before After Before After Before After

1.0

Typhe 0.8

/d r

m r

y

0.6

f

/

d

Monstera

la_

"~> 0.4 la_

0.2

0

I

0

I

I

I

I

I

I

60 120 180 240 300 360 420 Time o f f e r shoding, min

Fig. 9. Time course of the recovery of Fv/F~, 692 at a low PFD

in air following 3-h photoinhibitory treatments of a sun leaf of Typha latifolia and a shade leaf of Monstera deliciosa. The leaves had been treated at 2000 gmol.m-2.s -1, 2% Oz, 0% CO2 (Typha) and 1850 gmol-m-2"s-1, air (Monstera) m a t u r e sun l e a f o f Typha a n d a m a t u r e shade leaf o f Monstera is s h o w n in Fig. 9. T o achieve sufficient p h o t o i n h i b i t i o n in Typha these t r e a t m e n t s were m a d e in 2 % O2, 0 % CO2. T h e r e c o v e r y was followed after r e t u r n to n o r m a l air a n d a low P F D . I n the Typha l e a f r e c o v e r y was rapid, a n d c o m p l e t e r e c o v e r y was o b t a i n e d within 2 h, w h e r e a s in the Monstera leaf r e c o v e r y was very slow a n d only a small partial r e c o v e r y was o b t a i n e d after 7 h. R o u g h estimates o f the rate c o n s t a n t s for rec o v e r y in sun a n d s h a d e leaves o f five species are given in T a b l e 4. These rate c o n s t a n t s were estim a t e d as described b y G r e e r et al. (1986). (The smaller r e d u c t i o n in the Fv/FM, 692 ratio after the

Fo, 692

FM, 692

Fv/F~, 692

9.8 15.8 11.1 13.2 7.8 10.5 9.0 7.9 7.2 5.1 10.7 8.4

70.6 22.1 64.2 23.2 48.7 17.0 44.0 20.0 27.1 14.2 51.5 25.0

0.862 0.284 0.827 0.431 0.84t 0.399 0.796 0.605 0.735 0.644 0.792 0.664

p h o t o i n h i b i t o r y t r e a t m e n t s in the sun leaves, together with the fast recovery, m a d e it impossible to o b t a i n a c c u r a t e values for the rate c o n s t a n t s a n d the values p r e s e n t e d here for the sun leaves are estimates o f m i n i m u m rate constants). It is apparent, however, t h a t the rate c o n s t a n t s f o r recovery in m a t u r e sun leaves were m u c h greater t h a n in m a t u r e shade leaves. This difference is also evid e n t w h e n m a t u r e sun leaves are c o m p a r e d with m a t u r e shade leaves o f a single species (Hedera). It is n o t e w o r t h y , however, t h a t y o u n g developing shade leaves o f b o t h Hedera a n d Sehefflera exhibited a m u c h greater c a p a c i t y for r e c o v e r y t h a n did m a t u r e shade leaves o f the s a m e t w o species. In Hedera the rate c o n s t a n t s for recovery were similar in y o u n g d e v e l o p i n g sun a n d shade leaves. T h e r e c o v e r y rate was f o u n d to be influenced by two o t h e r factors: 1) in Typha a n d Glycine leaves r e c o v e r y was greatly s u p p r e s s e d b y 2 % O2, 0 % CO2, a n d 2) in m a t u r e shade leaves o f Hedera a n d monstera r e c o v e r y was m u c h slower after t r e a t m e n t s t h a t caused severe p h o t o i n h i b i t i o n t h a n after t r e a t m e n t s t h a t caused m o d e r a t e p h o t o i n h i b i tion. Discussion

Fluorescence quenching. High-light t r e a t m e n t s o f leaves i n v a r i a b l y resulted in a p r o n o u n c e d quenching o f FM, 692 a n d FM, 734 in all o f the species studied. A m u c h smaller b u t significant q u e n c h i n g o f Fo, 734 was also observed. FM, 734 was q u e n c h e d to a p p r o x i m a t e l y the s a m e degree as Fv, 692. These results are in a g r e e m e n t with previous studies on leaves (e.g. Powles a n d B j 6 r k m a n 1982; B j 6 r k m a n a n d Powles 1984; L u d l o w a n d

B. Demmig and O. Bj6rkman: Response of fluorescence and p h o t o n yield to excessive light

181

Table 4. Estimated rate constant for recovery of the Fv/FM, 692 ratio (77K) following photoinhibitory treatments in leaves of different species, growth light regimes and developmental stages. Recovery took place at a P F D of 30 to 100 gmol. m - z . s - ~ Species

G r o w t h light regime

Developmental status

Fv/FM, 692 before recovery

Rate constant ( x 10 3, min 1) Air

Glycine max

2% 02, 0% COz

Winter daylight Winter daylight

Mature Mature

0.680 0.656

>>16

Hedera canariensis

Summer daylight Shade Shade Summer daylight Shade

Mature b Mature c Mature Developing a Developing ~

0.673 0.432 0.099 0.582 0.537

~>29 5.6 0.1 54 50

Monstera delieiosa

Deep shade Deep shade

Mature Mature

0.727 0.314

5.0 1.2

Schefflera aetinophylla

Shade Shade

Mature Developing

0.643 0.378

3.5 58

Typha actinophylla

Summer daylight Summer daylight

Mature a Mature"

0.575 0.621

2.6

~>28 2.5

a Photoinhibitory treatment took place in 2% Oz, 0% COz b, o, d, e Leaf chlorophyll contents were, in this order: 68.9, 63.6, 17.3 and 20.4

Bj6rkman 1984; Ogren and C)quist 1984c). In the present study a pronounced quenching of FM, 692 and FM, 734 was obseved, even when the high-light treatment had only a small effect on the photon yield of 02 evolution or on the Fv/FM, 692 ratio. This finding is consistent with previous results obtained with sun leaves of Nerium oleander (Bj6rkman and Powles 1984). A striking result of the present study is the great variation in the response of Fo, 692 to highlight treatments. In some species, high-light treatment caused a marked increase in Fo, 692; in others it caused a pronounced decrease; and in some cases it caused no consistent change. Moreover, the time course of the change in Fo, 692 during the high-light treatment was often complex; for example Fo, 692 could decline at first and then rise. These results indicate that two processes were operating concurrently: one process causing a rise in Fo, 692 and the other causing a general quenching of fluorescence. For further discussion of this point it is useful to recall that simple theory predicts the following relationships (Kitajima and Butler 1975). In the state where all PSII traps are open, Fo, 692 = Kv/(Kv + KD + Kp) and in the state where all traps are closed FM, 692 = KF/(KF+ KD). KF and Kp denote the rate constants for fluorescence and photochemistry, respectively, and KD primarily denotes the rate constant for non-radiative (thermal) energy dissipation in the pigment bed but would also include any energy transfer from PSII to PSI. Any damage to the system which

decreases the rate constant for photochemistry (Kp) would increase PSII fluorescence in the state of open traps (Fo, 692) but, it would have no effect in the state of closed traps (FM, 692). In this case the decrease in Fv, 692 and in the Fv/FM, 692 ratio would therefore be caused by an increase in Fo, 692. The Fv/FM, 692 ratio would be directly proportional to the efficiency of photochemistry (Kitajima and Butler 1975). High-light treatments also cause an increase in KD. This would have an effect both in the state of open traps and in the state of closed traps. Hence, both Fo, 692 and FM, 692 would be quenched, although not to the same degree. As long as KD remains relatively small in relation to Kp, an increase in KD can result in a pronounced quenching of FM, 692 with only a small (and barely detectable) decrease in Fo, 692 and a small decrease in the Fv/FM, 692 ratio. As in the situation where photoinhibition causes a reduction in Kp the fall in Fv/FM, 692, caused by a rise in KD, would be directly proportional to the fall in the efficiency of primary photochemistry over a very wide range. An increase in KD caused by an increased transfer to PSI would result in an increased fluorescence emission at 734 nm. Since the high-light treatment caused a quenching rather than an increase of fluorescence at 734 nm it seems safe to conclude that the quenching of fluorescence at 692 nm was caused by an increased non-radiative energy dissipation. (Apparently, these treatments also caused an increase in the rate constant for non-radiative decay in PSI). Since such non-radia-

182

B. Demmig and O. Bj6rkman: Response of fluorescence and photon yield to excessive light

tive decay will divert excitation energy from the reaction centers, it could also provide some degree of protection from overexcitation of these centers. The responses of Fo, 692 and FM, 692 to treatments at 2% 02, 0% CO2 at a low P F D can be explained by an increase in non-radiative decay (KD). These experiments were conducted at a low P F D to avoid photoinhibitory damage. The treatments resulted in a general fluorescence quenching at both 692 and 734 nm. The quenching of Fo, 692 and FM, 692 observed here, closely resembles the effects observed by Kitajima and Butler (1975) upon addition of dibromothymoquinone to a chloroplast suspension. Our results are in agreement with those expected if the sole cause of the quenching were a rise in KD. The small but significant decline in the Fv/FM, 692 ratio and in the photon yield of O2 evolution, caused by 2% 02, 0% CO2 treatment, would be a direct consequence of an increase in KD. This means that an increase in the rate constant for non-radiative decay does indeed cause a decrease in the efficiency of primary photochemistry and therefore, could be considered a kind of photoinhibition. However, as long as this response is rapidly reversible, we do not consider it a reflection of photoinhibitory damage but rather a consequence of a regulatory process. Photoinhibitory damage is likely to result primarily from an inactivation of the PSII reaction center complex and affects fluorescence characteristics by decreasing Kp. Such photoinhibition does not appear to be rapidly reversible, and repair of such damage evidently involves chloroplast-encoded protein synthesis (Ohad et al. 1984; Greer et al. 1986).

Mechanism of the fluorescence quenching. We do not know what mechanism underlies the general quenching of fluorescence observed when leaves kept in weak light were treated at 2% Oz, 0% CO2. We assume that the same quenching mechanism operates when leaves are exposed to bright light in normal air. At least two kinds of mechanisms have been proposed that reversibly quench fluorescence when the excitation energy exceeds the rate which this energy can be used in photosynthesis. The first, termed energy quenching, is widely considered to be associated with a build-up of a ApH across the thylakoid membrane. However, the half-time for the relaxation of this quenching upon darkening has been determined to be only about 5 s in Chlorella cells and about 15 s in isolated spinach chloroplasts (Krause etal. 1982, 1983). The dark period preceding each of our fluorescence measurements was 300 s and the half-time of the relaxation for the quenching upon return

from 2% 02, 0 % C O 2 to air at a low PFD, or from a high to a low P F D in normal air, was in the order of 1 200-2 000 s. Moreover, while the rapid high-light-induced quenching of FM was abolished in the presence of 3 3,4-dichlorophenyl1,1-dimethylurea ( D C M U ) a slower quenching still occurred. Indeed, the slow quenching was more pronounced in DCMU-treated than in untreated leaves. On the assumption that the mechanism of the slow quenching seen in DCMU-treated leaves is the same as that in untreated leaves it appears that this quenching can occur even under conditions that should minimize a build-up of a ApH across the thylakoid membrane. However, the mechanisms of the quenching observed in the absence and presence of D C M U may not be identical. A second quenching mechanism is thought to be caused by phosphorylation of the light-harvesting chlorophyll-protein complex of PSII (LHC-II; for a review, see H o r t o n 1985). This process has a time constant more in line with that for the quenching observed here. However, the present quenching affected both F, 692 and F, 734 and therefore, cannot be explained by a State I-State II transition. More importantly, other experiments (Demming et al. 1987) indicate that this fluorescence quenching in soybean leaves was not accompanied by phosphorylation of the LHC-II; instead a dephosphorylation was observed. The time courses of the fluorescence quenching observed here when leaves, kept at a low P F D were exposed to high P F D as well as the relaxation of this quenching after return to a low PFD, closely resemble those of the high-light-induced accumulation of zeaxanthin and its reconversion to violaxanthin in weak light (Yamamoto 1979). This raises the possibility of a specific role of the violaxanthinzeaxanthin cycle in effecting the light-modulated changes in non-radiative energy dissipation in leaves.

Relationship between fluorescence character&tics and the photon yield of 02 evolution. As mentioned above, theory predicts that any reduction in the

Fv/FM, 692 ratio should be a quantitative indicator of the reduction in the efficiency of the primary photochemistry of PSII, irrespective of whether the reduction in theFv/FM, 692 ratio is caused by an increase in KD or by a decrease in Kp. Our results show that there is a close relationship between the Fv/FM, 692 ratio and the photon yield of 02 evolution in leaves of several species which has been subjected to various photoinhibitory treatments, both in the short term and in the longer term.

B. Demmig and O. Bj6rkman: Response of fluorescence and photon yield to excessive light

This provides experimental evidence for the validity of the theory. It should be noted that these measurements of photon yields and Fv/FM, 692 ratios were made after allowing for recovery in weak light from 1 to 7 h. Any rapidly reversible increases in KD should have subsided by that time. It should also be noted that the regression lines obtained when the photon yield is plotted as function of the Fv/FM, 692 ratio did not extrapolate to the origin. An even greater offset was obtained with Monstera deliciosa leaves, when shorter recovery times were used (data not presented). This may indicate that the photoinhibitory treatments had effects on photosynthesis in addition to those caused by inactivation of the primary photochemistry of PSII. The new information on the relationship between fluorescence characteristics and the photon yield of Oz evolution in photoinhibited leaves, gained in the present study, has implications on the quantitative aspects of photoinhibition in the previous studies by Bj6rkman and Powles (1984) and Ludlow and Bj6rkman (1984). However, reexamination of their results in view of the new information does not alter the basic conclusions put forward in these papers. Photoinhibition and recovery in sun and shade leaves. The present results are consistent with previous observations that leaves of plants which normally occupy sunny habitats (" sun species") and allowed to develop under high light are much less susceptible to photoinhibition than are shade leaves of species that are restricted to shaded habitats in nature (" shade species"). In the past the implicit assumption has been made that the lower susceptibility to photoinhibition in sun leaves than in shade leaves is a direct consequence of the greater light-saturated photosynthetic capacity of sun leaves, which allows a greater dissipation of excitation energy via photosynthesis itself (Bj6rkman 1981; Powles 1984). However, the present results indicate that other factors are also involved. Measurements of the Fv/ FM, 692 ratio showed that sun leaves still exhibited less photoinhibitory damage than shade leaves, even when exposed to bright light under conditions that prevented photosynthesis (2% O2, 0% CO2). In addition the rate of recovery in air that was observed after photojnhibited leaves were returned to a low-light regime was much faster in mature sun leaves than in mature shade leaves. Such recovery from photoinhibition has previously been shown to be light and temperature dependent (Greer et al. 1986) and probably requires chloroplast-encoded protein synthesis

183

(Ohad et al. 1984). It therefore appears that the recovery is caused by a repair process which presumably operates both during and after the highlight treatments. Thus, in leaves with a high capacity for repair, the net photoinhibition observed after a given treatment would be smaller than in a leaf with a low capacity for repair, even if the inhibition process itself proceeded with the same rate in the two kinds of leaves. The present results indicate that differences in the capacity for repair can be an important contributing factor in the observed differences in susceptibility to photoinhibition between mature sun and mature shade leaves. However, the capacity for repair also seems to depend on the developmental stage of the leaf, being greater in young developing shade leaves than in mature shade leaves. One may speculate that this difference is related to a higher rate of protein synthesis in developing leaves, Recovery at a low P F D was greatly suppressed by 2% 0 2 , 0 % C O 2 in Typha latifolia leaves, indicating that the process required some degree of photosynthetic activity. Such a requirement could explain the observation that the recovery rate in air was lower in leaves that had suffered severe photoinhibitory damage than in leaves in which photoinhibition was moderate. We thank Karen Hall and Connie Shih for providing technical assistance and Max Seyfried and Engelbert Weis for reading the manuscript. Post-doctoral support from the Deutsche Forschungsgemeinschaft, the McKnight Foundation and the Carnegie Corporation of New York to B.D. is gratefully acknowledged.

References Bj6rkman, 0. (1981) Responses to different quantum flux densities. In: Encyclopedia of Plant Physiology, N.S,, vol. 12 A : Interactions with the physical environment, pp. 57-107. Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer-Verlag, Berlin Heidelberg New York Bj6rkman, 0., Boardman, N.K., Anderson, J.M., Thorne, S.W., Goodchild, D.J., Pylotis, N.A. (1972) Effect of light intensity during growth of Atriplex patula on the capacity of photosynthetic reactions, chloroplast components and structure. Carnegie Inst. Washington Yearb. 71, 115-135 Bj6rkman, O., Demmig B. (1987) Photon yield of 0 2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origin. Planta 170, 489-504 Bj6rkman, O., Powles, S.T. (1984) Inhibition of photosynthetic reactions under water stress: interaction with light level. Planta 161, 49(~504 Butler, W.L. (1978) Energy distribution in the photochemical apparatus of photosyntheis. Annu. Rev. Plant Physiol. 29, 345-378 Critchley, C., Smillie, R.M. (1981) Leaf chlorophyll fluorescence as an indicator of photoinhibition in Cucumis sativus L. Aust. J. Plant Physiol. 8, 133-141 Demmig, B., Cleland, R., Bj6rkman, O. (1987) Photoinhibition,

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B. Demmig and O. Bj6rkman: Response of fluorescence and photon yield to excessive light

chlorophyll fluorescence (77K) and phosphorylation of LHC-II. Planta 111, in press Ehleringer, J., Bj6rkman, O. (1977) Quantum yields for CO2 uptake in C3 and C4 plants. Dependence on temperature, CO2 and 02 concentrations. Plant Physiol. 59, 86-90 Fork, D.C., Oquist, G., Powles, S.B. (1981) Photoinhibition in bean: A fluorescence analysis. Carnegie Inst. Washington Yearb. 80, 52-57 Greer, D., Berry, J.A., 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. (1985) Interactions between electron transfer and carbon assimilation. In: Photosynthetic mechanisms and the environment, vol. 6, pp. 135-187, Barber, J., Baker, N.R., eds. Elsevier Biomedical Press, Amsterdam Kitajima, M., Butler, W.L. (1975) Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim. Biophys. Acta 376, 105 115 Krause, G.H., Briantais, J.-M., Vernotte, C. (t983) Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77K. I. ApH-dependent quenching. Biochim. Biophys. Acta 723, 169-175 Krause, G.H., Vernotte, C., Briantais, J.-M. (1982) Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components. Biochim. Biophys. Acta 679, 116-124 Ludlow, M., Bj6rkman, O. (1984) Paraheliotropic leaf movement in Siratro as a protective mechanism against droughtinduced damage to primary photosynthetic reactions: damage by excessive light and heat. Planta 161, 505-518 Ogren, E., Oquist, G. (1984a) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. I. Photosynthesis in vivo. Physiol. Plant. 62, 181-186

Ogren, 12., Oquist, G. (1984b) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. II. Photosynthetic electron transport. Physiol Plant. 62, 187 192 Ogren, 12., Oquist, G. (1984c) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77 K. Physiol. Plant. 62, 193-200 Ohad, I., Kyle, D.J., Arntzen, C.J. (1984) Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes. J. Cell. Biol. 99, 481485 Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 15-44 Powles, S.B., Bj6rkman, O. (1981) Leaf movement in the shade species Oxalis oregana. II. Role in protection against injury by intense light. Carnegie Inst. Washington Yearb. 80, 63-66 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photosynthesis : effect on chlorophyll fluorescence at 77 K in intact leaves and in chloroplast membranes. Planta 156, 97-107 Powles, S.B., Osmond, C.B., Thorne, S.W. (1979) Photonhibition of intact attached leaves of C3 plants illuminated in the absence of both carbon dioxide and of photorespiration. Plant Physiol. 64, 982-988 Schreiber, U., Schliwa, U., Bilger, W. (1986) Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Res. 10, 51-62 Yamamoto, H.Y. (1979) Biochemistry of the violaxanthin cycle in higher plants. Pure Appl. Chem. 51,639-648

Received 20 June 1986; accepted 12 February 1987