Photosynthesis Research 10:303-308 (1986) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
303
ENERGY-DEPENDENT QUENCHING OF DARK-LEVEL CHLOROPHYLL FLUORESCENCE IN INTACT LEAVES. W. BILGER AND U.
SCHREIBER
Institut f o r B o t a n i k und P h a r m a z e u t i s c h e B i o l o g i e der Universit ~ t WOrzburg, M i t t l e r e r D a l l e n b e r g w e g 64, D-8700 WOrzburg, FRG
ABSTRACT A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (qE) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of C02) not only variable fluorescence, Fv, but also darklevel fluorescence, FO, was quenched, leading to definition of a quenching coefficient, qo" Information on qo was shown to be essential for correct determination of photochemical (qQ) and energy dependent quenching (qE) by the saturation pulse method. The relationship between qE and qo was analysed over a range of light intensities at steady state conditions, qE was found to consist of two components, the second of which is linearly correlated with qo" qo and the second component of qE are interpreted to reflect the state 1 - state 2 shift caused by LHC II phosphorylation.
INTRODUCTION Various quenching mechanisms determine c h l o r o p h y l l fluorescence y i e l d i n v i v o ( f o r recent reviews, see r e f s . 22, 21, 11, 25). Major quenching components are Q-quenching (qQ), due to photochemical energy conversion at PS I I r e a c t i o n centers, and energy-dependent quenching (qE), due to an i n creased r a t e of r a d i a t i o n l e s s d e e x c i t a t i o n upon " e n e r g i z a t i o n " of the t h y l a k o i d membrane (22, 10, 8, 19) D i f f e r e n t i a t i o n between qD and qE i s required f o r the i n t e r p r e t a t i o n of the complex fluorescence ~nduction k i n e t i c s (Kautsky e f f e c t ) , which provides important i n f o r m a t i o n on the s t a t e of the photosynthetic apparatus. Recently, there has been considerable progress in the attempt to d i f f e r e n t i a t e between qQ and qE (19, 8, 9, 23, 24, 14, 26, 27). By applying short pulses of s a t u r a t i n g l i g h t , i t i s possible to momentarily remove qQ. I t has been assumed t h a t any remaining quenching p r i m a r i l y r e f l e c t s qE , (9, 23, 14, 26). For the c a l c u l a t i o n of qo and qE, the basic assumption has been made that only variable fluorescence-(F v) is affected by q~. For qQ = 1, i.e., at the Fo-level, no energy-dependent quenching ha~ been assumed. In the present report it is shown that suppression of F 0 may occur under conditions of strong energization. The relationship between energy-dependent quenching of F v, (qE), and of FO, (qo), is analysed. With this information on qo it is possible to derive more accurate values of qQ and qE. Furthermore, the properties of qo suggest that it may be an indicator for state 1 - state 2 shifts i__n vivo, due to LHC II phosphorylation (5, 16, 1, 15). MATERIALS AND METHODS Experiments were c a r r i e d out with detached leaves of Arbutus unedo and
[1571
304
[1581
of a number of other plants, grown at the Institute's Botanical Garden. Leaf samples, the petiole of which was immersed in water, were exposed to a stream of water-saturated air (30 l x h -1) with or without CO 2. Chlorophyll fluorescence was measured with a new type of modulation fluorometer (26, 27), which is now commercially available (PAN 101 Chlorophyll Fluorometer; H. Walz, Effeltrich, Germany). This system monitors fluorescence yield with a weak modulated measuring beam (integrated intensity 1 mW x m-2). Selective amplification of the modulated signal is not disturbed by actinic illumination and application of saturation pulses. Light intensities were measured with a LICOR 185 B Radiometer. R E S U L T S AND D I S C U S S I O N Fig. 1 shows chlorophyll fluorescence induction curves of an Arbutus leaf in the presence and absence of CO 2 monitored by a pulse-modulated measuring beam (26, 27). In addition to continuous actinic illumination, there is repetitive application (at 0.1 Hz) of short saturation pulses to differentiate between redox-dependent quenching (qQ) and energy-dependent quenching (qE). Following a rapid build-up of qE, only the sample in the presence of CO 2 shows relaxation of qE, as may be expected from the onset of Calvin cycle activity (24, 26). When actinic illumination is switched off, there is a substantial transient decrease of fluorescence below the original FOlevel in the sample without CO 2 (see also inset of Fig. 1). In this sample, steady state fluorescence also drops below the original FO. The occurrence of Fo-quenching has to be taken into account when the quenching coefficients, qQ and qE, are calculated. In Fig. 2 the definition of a new quenching coefficient, qo, and the consequences for qQ and qE determinations are depicted (for comparison see Fig. 3 in ref. 26). The assumption is made that Fo-quenching reflects a decrease of absorbed energy directed to PS II, formally equivalent to a decrease in excitation light intensity. Hence, besides FO, also variable fluorescence is correspondingly suppres-
a) U
-COz
• C02
,-5
F~
4
L_
o
~3
LL 2 Fo
O
'on t rn.b.
FIGURE 1.
~off 2 w i n -4
0
Tm.b.
Energy d e p e n d e n t q u e n c h i n g o f Fv and F0 i n r e l a t i o n
t o CO2
availability in intact leaves of Arbutus unedo. Actinic intensity, 60 W/m 2. Saturation pulses, 1000 W/m 2 for 700 ms. The arrows indicate switching on of the measuring beam ( m.b.), application of a saturation pulse ( ~ ) for determination of Fm and switching on/off of the actinic light ( ~ ) . In the (-C02) experiment, the light-off response is also shown at 8-fold amplification in the inset.
[159]
305
F~ = Fo- Fo. qo
(1-qo}=
8
F'
F=' F0
=F;*
(1)
(21
F,' = Fo t l - q o ) *
o
*(Fv} m • (1-qo)(I-qo)(1-qEJ ~t
(Fv) j"
~
( 3)
..........
0
. . . . I m.b.
Time
= ( F v ) e n . l l - q o ) ( t - q E)
(4)
(~-%1 =
Fv" (Fv),'
(5)
(1- Cle) =
(F~,)=" IFv)m,(1-q o)
(6)
FIGURE 2. Definition of quenching coefficients under conditions of FO-quenching. Schematic reproduction of an experimental curve equivalent to that of Fig. 1 (-C02) the proportions of which were changed for illustration. See text for further explanation.
sed. As revealed by equations (5) and (6), the existence of Fo-quenching affects qQ and qE-determinations in two ways; First, the new Fo'-level has to be taken into account for evaluation Fv' and (Fv)s'. Second, the maximal possible variable fluorescence (for qQ = 0 and qE = 0), which is not directly accessible, must be assumed to be (Fv)m' = (Fv)m (1 - qo). Making use of equations (2), (5) and (6) (see Fig. 2), the different quenching coefficients may be calculated for the experiment of Fig. 1. Shortly before the actinic light was switched off, with CO 2 present, qo = qQ = 0.91 and qE = 0.20; in absence of CO 2, qo = 0.23, qQ = 0.83 and qE = 0.83. In this example, ignorance of Fo-quenching would have led to an overestimation of qE by about 15%; no meaningful value for qQ could have been calculated with Fv going negative. We have observed Fo-quenching in leaves of a large variety of different plants, under conditions which also produce strong energy dependent quenching of F v. So far, there appears to be an upperlimit of qo = 0.35. In certain species, like Phaseolus vulqaris and Vicia faba, steady state fluorescence was well above FO, and no Fo-quenching could be detected, even when strong energy quenching was induced. The relationship between qE, qQ and qo in Arbutus unedo is shown in Fig. 3 A, B for steady state conditions at different light intensities in the absence of CO 2. It is apparent that substantial qE accumulated before any qo was detected. The increase in qE consisted of two distinct components, and the second component corresponded with the increase in qo. The decrease in qQ exhibited three successive transitions with increasing amplitude. In Fig. 3 B a plot of qo versus qE is presented, which suggests a linear dependency between the second component of qE and qo. When this dependency was extrapolated to qo = O, the separated first component of qE amounted to 0.5.
[1601
306
1.o r
tO
rO) 0
10
i
B.
A. -
~
±-
qE
0.8
08
0.6 (..)
06
o u~ 0.4
0.4
%
t-
0
0.2
I.L 0.2
50
lOO
15o
200
Light irrtensity, Wm -2
I 250
0,0
J
1
0.2
i
0,3
0.0
q0
FIGURE 3. L i g h t i n t e n s i t y dependency o f the quenching c o e f f i c i e n t s i n Arbutus unedo, i n absence o f CO2. ~ Plot o l i fg hqQ,t qE, and qo versus l i g h t i n t e n s i t y . B. P l o t of qEversus qo" each i n t e n s i t y , quenching was evaluated at s t e a d y - s t a t e i l l u m i n a t i o n (about 5-min e q u i l i b r a t i o n between successive l i g h t i n t e n s i t i e s ) :
We have considered the p o s s i b i l i t y t h a t qo i n our measurements might be an a r t i f a c t r e s u l t i n g from changes i n e f f e c t i v e l i g h t absorption or f l u orescence r e a b s o r p t i o n , accompanying l i g h t - i n d u c e d changes i n o p t i c a l prop e r t i e s of the leaves. Indeed, t h e r e i s close c o r r e l a t i o n between FOquenching and the increase o f apparent absorbance o f green l i g h t ( " l i g h t s c a t t e r i n g " ) ( B i l g e r , Heber and Schreiber, manuscript i n p r e p a r a t i o n ) . However, we do not consider qo t o be simply a m a n i f e s t a t i o n o f a l t e r e d l e a f absorption f o r the f o l l o w i n g reasons: F i r s t , f l u o r e s c e n c e was measured from the l e a f surface and the 650 nm measuring l i g h t can be assumed t o be s t r o n g l y absorbed i n the surface l a y e r o f l e a f c h l o r o p l a s t s . I f l e a f o p t i cal changes would mimic f l u o r e s c e n c e quenching, t h i s should be s u b s t a n t i a l l y enhanced i f f l u o r e s c e n c e i s measured through the l e a f . In r e a l i t y , qo was found to be identical when fluorescence was measured from the surface or through the leaf (not shown in the figures). Second, large increases in "light scattering" were induced in the dark by changes in leaf water content without observation of any effect on FO, measured in the same way as in the experiments described above (not shown in the figures). Hence, the observed changes of qo should be considered real. The tripartite model of Butler (12, 13, 28), as well as recent insights into the topology of the thylakoid membrane (2 - 4) and into state 1 state 2 shifts by LHC II phosphorylation (5, 16, 15, 17, 20) may provide satisfactory interpretations for the presented results. According to Butler (12, 13), F0 is determined by the extent of PS II excitation when all PS II traps are open. A decrease of F0 (at conditions of constant light absorbance) does suggest a decrease of energy transfer from the LHC II to PS II, which was found to take place upon LHC II phosphorylation (15, 20). There is lateral movement of this pigment complex away from high fluorescence PS II in the partition region towards low fluorescence P S I in the margin
[161l
307
r e g i o n of the t h y l a k o i d s . I n i t i a t i o n o f p r o t e i n p h o s p h o r y l a t i o n r e q u i r e s r e d u c t i o n of the plastoquinone pool (6, 18, 29). Indeed, the data o f Fig. 3 confirm t h a t development o f qo i s retarded u n t i l qQ begins to d e c l i n e . In t h i s c o n t e x t , i t should be considered, t h a t a small amount o f QA- ( c o r r e s ponding to qQ = 0.97) i s i n e q u i l i b r i u m w i t h reduced secondary acceptor QB- which w i l l accumulate even i n presence of an o x i d i z e d PQ-pool (7, 30, 31). F u r t h e r decrease i n qQ r e f l e c t s the accumulation of centers i n the s t a t e QA- QB= when r e o x i d a t i o n i s prevented due t o r e d u c t i o n o f the PQpool. The p o s t u l a t e d s t a t e s h i f t r e s u l t s i n s t i m u l a t e d P S I a c t i v i t y and reduced PS I I a c t i v i t y , r e l e a s i n g e l e c t r o n pressure on PQ and p o s s i b l y causing the i n f l e c t i o n i n the l i g h t - i n t e n s i t y dependency o f qQ (Fig. 3 A) around 70 W/m ~. A c o r r e l a t i v e study o f Fo-quenching and low temperature f l u o r e s c e n c e spectroscopy w i l l be r e q u i r e d to determine whether or not t h i s i n t e r p r e t a t i o n of qo by LHC I t p h o s p h o r y l a t i o n can be s u b s t a n t i a t e d . In c o n c l u s i o n , changes i n qo and qE, which have become r e a d i l y a c c e s s i b l e by the new modulation f l u o r o m e t e r , may provide a means of assessing energy dependent s t a t e s h i f t s i n i n t a c t leaves. Such s t a t e s h i f t s play an i m p o r t ant r o l e i n the r e g u l a t i o n of p h o t o s y n t h e s i s . ACKNOWLEDGEMENTS We wish to thank U l r i c h Heber f o r s t i m u l a t i n g d i s c u s s i o n s . Support by the Deutsche Forschungsgemeinschaft i s g r a t e f u l l y acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
A l l e n JF and Bennett J (1983) FEBS L e t t 1 2 3 : 6 7 - 7 0 Andersson B and Anderson JM (1980) Biochim Biophys Acta 5 9 3 : 4 2 7 - 4 4 0 Anderson JM (1981) FEBS L e t t 1 2 4 : 1 - 1 0 Barber J and Chow WS (1979) FEBS L e t t 1 0 5 : 5 - 1 0 Bennett J, Steinback KE and Arntzen CJ (1980) Proc Natl Acad Sci USA 77:5253-5257 Bennett J (1983) Biochem J 2 1 2 : 1 - 1 3 Bouges-Boequet B (1984) Biochim Biophys Acta 3 1 4 : 2 5 0 - 2 5 6 Bradbury M and Baker NR (1981) Biochim Biophys Acta 6 3 5 : 5 4 2 - 5 5 1 Bradbury M and Baker NR (1984) Biochim Biophys Acta 7 6 5 : 2 7 5 - 2 8 1 B r i a n t a i s JM, Vernotte C, Picaud M and Krause GH (1980) Biochim Biophys Acta 5 9 1 : 1 9 8 - 2 0 2 B r i a n t a i s JM, Vernotte C, Krause GH and Weis E (1986) In L i g h t Emission by Plants and B a c t e r i a (Govindjee, Amesz J and Fork DC, eds.) Academic Press, i n press, New York B u t l e r WL and K i t a j i m a M (1975) Proc 3rd Congr Photosynth, Vol. I , pp. 13-24 B u t l e r WL (1977) In Encyclopedia o f P l a n t Physiology (Trebst A and Avron M eds.) Vol. V, pp. 149-167, Springer Verlag, Heidelberg D i e t z KJ, Schreiber U and Heber U (1985) Planta 1 6 6 : 2 1 9 - 2 2 6 Hodges M and Barber J (1983) Plant P h y s i o l 7 2 : 1 1 1 9 - 1 1 2 2 Horton P and Black MT (1980) FEBS L e t t 1 1 9 : 1 4 1 - 1 4 4 Horton P and Black MT (1981) Biochim Biophys Acta 6 3 5 : 5 3 - 8 2 Horton P, A l l e n JF, Black MT and Bennett J (1981) FEBS L e t t 125: 193-196 Krause GH, B r i a n t a i s JM and Vernotte C (1983) Biochim Biophys Acta 723:169-175 Krause GH, Behrend U (1983) Biochim Biophys Acta 7 2 3 : 1 7 6 - 1 8 1 Krause GH and Weis E (1984) Photosynth Res 5 : 1 3 9 - 1 5 7 Lavorel J and Etienne AL (1977) In Primary Processes o f Photosynthesis (Barber J, ed.) pp. 202-268 E l s e v i e r , Amsterdam
308
[162l
23. Quick P and Horton P (1984) Proc R Soc Lond 2 2 0 : 3 6 1 - 3 7 0 24. Quick P and Horton P (1984) Proc R Soc Lond 2 2 0 : 3 7 1 - 3 8 2 25. Renger G and Schreiber U (1986) In Light Emission by Plants and Bacteria (Govindjee, Amesz d and Pork DC, eds.) Academic Press, in press, New York 26. Schreiber U, Bilger W and Schliwa U (1985) Photosynth 8es, in press 27. Schreiber U (1986) Special Issue of Photosynth Res, in press 28. Strasser RJ and Butler WL (1977) Biochim Biophys Acta 462:295-306 29. Teller A, Bottin H, Barber d and Mathis P (1984) Biochim Biophys Acta 764:324-330 30. T h i e l e n APG and van Gorkom HJ (1981) FEBS L e t t 1 2 9 : 5 7 - 6 1 31. Velthuys BR and Amesz J (1974) Biochim Biophys Acta 3 3 3 : 8 5 - 9 4 Note added a f t e r manuscript submission: Very r e c e n t l y Malkin, T e l l e r and Barber (Biochim. Biophys. Acta 848, 48-. 57, 1986) reported on fluorescence measurements with a conventional modulation technique, suggesting changes in absorption cross-section of the two photosystems accompanying changes in F 0 and F v during Light 1 - Light 2 induced state changes. While our findings do agree with the conclusions of these authors, it should be noted that there is a principal difference in the definition of F 0 in the approach of Malkin and co-workers as compared to our study. These authors determine "Fo" using a measuring light of 7 W/m 2 which is 7000 times stronger than what was applied here, and which is sufficiently intensive to induce appreciable variable fluorescence. Hence, the observed changes in "Fo" could well represent, at least in part, changes in Fv.Even if,as suggested by Malkin (Isr. d. Chem. 21, 306-315, 1981), the F v which can not be quenched by Light 1 originated from "unconnected PS II centers", it remains uncertain to what extent the relative size of this population of centers may vary during state changes. Such variation would cause changes in F v mimicking changes in "Fo". Furthermore, besides of non-photochemical quenching induced by a state 1 - state 2 transition, also changes due to membrane energization (qE) should be considered. Therefore, we believe that for definite demonstration of true changes in absorption cross-section the actual F0 has to be measured.
303
ENERGY-DEPENDENT QUENCHING OF DARK-LEVEL CHLOROPHYLL FLUORESCENCE IN INTACT LEAVES. W. BILGER AND U.
SCHREIBER
Institut f o r B o t a n i k und P h a r m a z e u t i s c h e B i o l o g i e der Universit ~ t WOrzburg, M i t t l e r e r D a l l e n b e r g w e g 64, D-8700 WOrzburg, FRG
ABSTRACT A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (qE) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of C02) not only variable fluorescence, Fv, but also darklevel fluorescence, FO, was quenched, leading to definition of a quenching coefficient, qo" Information on qo was shown to be essential for correct determination of photochemical (qQ) and energy dependent quenching (qE) by the saturation pulse method. The relationship between qE and qo was analysed over a range of light intensities at steady state conditions, qE was found to consist of two components, the second of which is linearly correlated with qo" qo and the second component of qE are interpreted to reflect the state 1 - state 2 shift caused by LHC II phosphorylation.
INTRODUCTION Various quenching mechanisms determine c h l o r o p h y l l fluorescence y i e l d i n v i v o ( f o r recent reviews, see r e f s . 22, 21, 11, 25). Major quenching components are Q-quenching (qQ), due to photochemical energy conversion at PS I I r e a c t i o n centers, and energy-dependent quenching (qE), due to an i n creased r a t e of r a d i a t i o n l e s s d e e x c i t a t i o n upon " e n e r g i z a t i o n " of the t h y l a k o i d membrane (22, 10, 8, 19) D i f f e r e n t i a t i o n between qD and qE i s required f o r the i n t e r p r e t a t i o n of the complex fluorescence ~nduction k i n e t i c s (Kautsky e f f e c t ) , which provides important i n f o r m a t i o n on the s t a t e of the photosynthetic apparatus. Recently, there has been considerable progress in the attempt to d i f f e r e n t i a t e between qQ and qE (19, 8, 9, 23, 24, 14, 26, 27). By applying short pulses of s a t u r a t i n g l i g h t , i t i s possible to momentarily remove qQ. I t has been assumed t h a t any remaining quenching p r i m a r i l y r e f l e c t s qE , (9, 23, 14, 26). For the c a l c u l a t i o n of qo and qE, the basic assumption has been made that only variable fluorescence-(F v) is affected by q~. For qQ = 1, i.e., at the Fo-level, no energy-dependent quenching ha~ been assumed. In the present report it is shown that suppression of F 0 may occur under conditions of strong energization. The relationship between energy-dependent quenching of F v, (qE), and of FO, (qo), is analysed. With this information on qo it is possible to derive more accurate values of qQ and qE. Furthermore, the properties of qo suggest that it may be an indicator for state 1 - state 2 shifts i__n vivo, due to LHC II phosphorylation (5, 16, 1, 15). MATERIALS AND METHODS Experiments were c a r r i e d out with detached leaves of Arbutus unedo and
[1571
304
[1581
of a number of other plants, grown at the Institute's Botanical Garden. Leaf samples, the petiole of which was immersed in water, were exposed to a stream of water-saturated air (30 l x h -1) with or without CO 2. Chlorophyll fluorescence was measured with a new type of modulation fluorometer (26, 27), which is now commercially available (PAN 101 Chlorophyll Fluorometer; H. Walz, Effeltrich, Germany). This system monitors fluorescence yield with a weak modulated measuring beam (integrated intensity 1 mW x m-2). Selective amplification of the modulated signal is not disturbed by actinic illumination and application of saturation pulses. Light intensities were measured with a LICOR 185 B Radiometer. R E S U L T S AND D I S C U S S I O N Fig. 1 shows chlorophyll fluorescence induction curves of an Arbutus leaf in the presence and absence of CO 2 monitored by a pulse-modulated measuring beam (26, 27). In addition to continuous actinic illumination, there is repetitive application (at 0.1 Hz) of short saturation pulses to differentiate between redox-dependent quenching (qQ) and energy-dependent quenching (qE). Following a rapid build-up of qE, only the sample in the presence of CO 2 shows relaxation of qE, as may be expected from the onset of Calvin cycle activity (24, 26). When actinic illumination is switched off, there is a substantial transient decrease of fluorescence below the original FOlevel in the sample without CO 2 (see also inset of Fig. 1). In this sample, steady state fluorescence also drops below the original FO. The occurrence of Fo-quenching has to be taken into account when the quenching coefficients, qQ and qE, are calculated. In Fig. 2 the definition of a new quenching coefficient, qo, and the consequences for qQ and qE determinations are depicted (for comparison see Fig. 3 in ref. 26). The assumption is made that Fo-quenching reflects a decrease of absorbed energy directed to PS II, formally equivalent to a decrease in excitation light intensity. Hence, besides FO, also variable fluorescence is correspondingly suppres-
a) U
-COz
• C02
,-5
F~
4
L_
o
~3
LL 2 Fo
O
'on t rn.b.
FIGURE 1.
~off 2 w i n -4
0
Tm.b.
Energy d e p e n d e n t q u e n c h i n g o f Fv and F0 i n r e l a t i o n
t o CO2
availability in intact leaves of Arbutus unedo. Actinic intensity, 60 W/m 2. Saturation pulses, 1000 W/m 2 for 700 ms. The arrows indicate switching on of the measuring beam ( m.b.), application of a saturation pulse ( ~ ) for determination of Fm and switching on/off of the actinic light ( ~ ) . In the (-C02) experiment, the light-off response is also shown at 8-fold amplification in the inset.
[159]
305
F~ = Fo- Fo. qo
(1-qo}=
8
F'
F=' F0
=F;*
(1)
(21
F,' = Fo t l - q o ) *
o
*(Fv} m • (1-qo)(I-qo)(1-qEJ ~t
(Fv) j"
~
( 3)
..........
0
. . . . I m.b.
Time
= ( F v ) e n . l l - q o ) ( t - q E)
(4)
(~-%1 =
Fv" (Fv),'
(5)
(1- Cle) =
(F~,)=" IFv)m,(1-q o)
(6)
FIGURE 2. Definition of quenching coefficients under conditions of FO-quenching. Schematic reproduction of an experimental curve equivalent to that of Fig. 1 (-C02) the proportions of which were changed for illustration. See text for further explanation.
sed. As revealed by equations (5) and (6), the existence of Fo-quenching affects qQ and qE-determinations in two ways; First, the new Fo'-level has to be taken into account for evaluation Fv' and (Fv)s'. Second, the maximal possible variable fluorescence (for qQ = 0 and qE = 0), which is not directly accessible, must be assumed to be (Fv)m' = (Fv)m (1 - qo). Making use of equations (2), (5) and (6) (see Fig. 2), the different quenching coefficients may be calculated for the experiment of Fig. 1. Shortly before the actinic light was switched off, with CO 2 present, qo = qQ = 0.91 and qE = 0.20; in absence of CO 2, qo = 0.23, qQ = 0.83 and qE = 0.83. In this example, ignorance of Fo-quenching would have led to an overestimation of qE by about 15%; no meaningful value for qQ could have been calculated with Fv going negative. We have observed Fo-quenching in leaves of a large variety of different plants, under conditions which also produce strong energy dependent quenching of F v. So far, there appears to be an upperlimit of qo = 0.35. In certain species, like Phaseolus vulqaris and Vicia faba, steady state fluorescence was well above FO, and no Fo-quenching could be detected, even when strong energy quenching was induced. The relationship between qE, qQ and qo in Arbutus unedo is shown in Fig. 3 A, B for steady state conditions at different light intensities in the absence of CO 2. It is apparent that substantial qE accumulated before any qo was detected. The increase in qE consisted of two distinct components, and the second component corresponded with the increase in qo. The decrease in qQ exhibited three successive transitions with increasing amplitude. In Fig. 3 B a plot of qo versus qE is presented, which suggests a linear dependency between the second component of qE and qo. When this dependency was extrapolated to qo = O, the separated first component of qE amounted to 0.5.
[1601
306
1.o r
tO
rO) 0
10
i
B.
A. -
~
±-
qE
0.8
08
0.6 (..)
06
o u~ 0.4
0.4
%
t-
0
0.2
I.L 0.2
50
lOO
15o
200
Light irrtensity, Wm -2
I 250
0,0
J
1
0.2
i
0,3
0.0
q0
FIGURE 3. L i g h t i n t e n s i t y dependency o f the quenching c o e f f i c i e n t s i n Arbutus unedo, i n absence o f CO2. ~ Plot o l i fg hqQ,t qE, and qo versus l i g h t i n t e n s i t y . B. P l o t of qEversus qo" each i n t e n s i t y , quenching was evaluated at s t e a d y - s t a t e i l l u m i n a t i o n (about 5-min e q u i l i b r a t i o n between successive l i g h t i n t e n s i t i e s ) :
We have considered the p o s s i b i l i t y t h a t qo i n our measurements might be an a r t i f a c t r e s u l t i n g from changes i n e f f e c t i v e l i g h t absorption or f l u orescence r e a b s o r p t i o n , accompanying l i g h t - i n d u c e d changes i n o p t i c a l prop e r t i e s of the leaves. Indeed, t h e r e i s close c o r r e l a t i o n between FOquenching and the increase o f apparent absorbance o f green l i g h t ( " l i g h t s c a t t e r i n g " ) ( B i l g e r , Heber and Schreiber, manuscript i n p r e p a r a t i o n ) . However, we do not consider qo t o be simply a m a n i f e s t a t i o n o f a l t e r e d l e a f absorption f o r the f o l l o w i n g reasons: F i r s t , f l u o r e s c e n c e was measured from the l e a f surface and the 650 nm measuring l i g h t can be assumed t o be s t r o n g l y absorbed i n the surface l a y e r o f l e a f c h l o r o p l a s t s . I f l e a f o p t i cal changes would mimic f l u o r e s c e n c e quenching, t h i s should be s u b s t a n t i a l l y enhanced i f f l u o r e s c e n c e i s measured through the l e a f . In r e a l i t y , qo was found to be identical when fluorescence was measured from the surface or through the leaf (not shown in the figures). Second, large increases in "light scattering" were induced in the dark by changes in leaf water content without observation of any effect on FO, measured in the same way as in the experiments described above (not shown in the figures). Hence, the observed changes of qo should be considered real. The tripartite model of Butler (12, 13, 28), as well as recent insights into the topology of the thylakoid membrane (2 - 4) and into state 1 state 2 shifts by LHC II phosphorylation (5, 16, 15, 17, 20) may provide satisfactory interpretations for the presented results. According to Butler (12, 13), F0 is determined by the extent of PS II excitation when all PS II traps are open. A decrease of F0 (at conditions of constant light absorbance) does suggest a decrease of energy transfer from the LHC II to PS II, which was found to take place upon LHC II phosphorylation (15, 20). There is lateral movement of this pigment complex away from high fluorescence PS II in the partition region towards low fluorescence P S I in the margin
[161l
307
r e g i o n of the t h y l a k o i d s . I n i t i a t i o n o f p r o t e i n p h o s p h o r y l a t i o n r e q u i r e s r e d u c t i o n of the plastoquinone pool (6, 18, 29). Indeed, the data o f Fig. 3 confirm t h a t development o f qo i s retarded u n t i l qQ begins to d e c l i n e . In t h i s c o n t e x t , i t should be considered, t h a t a small amount o f QA- ( c o r r e s ponding to qQ = 0.97) i s i n e q u i l i b r i u m w i t h reduced secondary acceptor QB- which w i l l accumulate even i n presence of an o x i d i z e d PQ-pool (7, 30, 31). F u r t h e r decrease i n qQ r e f l e c t s the accumulation of centers i n the s t a t e QA- QB= when r e o x i d a t i o n i s prevented due t o r e d u c t i o n o f the PQpool. The p o s t u l a t e d s t a t e s h i f t r e s u l t s i n s t i m u l a t e d P S I a c t i v i t y and reduced PS I I a c t i v i t y , r e l e a s i n g e l e c t r o n pressure on PQ and p o s s i b l y causing the i n f l e c t i o n i n the l i g h t - i n t e n s i t y dependency o f qQ (Fig. 3 A) around 70 W/m ~. A c o r r e l a t i v e study o f Fo-quenching and low temperature f l u o r e s c e n c e spectroscopy w i l l be r e q u i r e d to determine whether or not t h i s i n t e r p r e t a t i o n of qo by LHC I t p h o s p h o r y l a t i o n can be s u b s t a n t i a t e d . In c o n c l u s i o n , changes i n qo and qE, which have become r e a d i l y a c c e s s i b l e by the new modulation f l u o r o m e t e r , may provide a means of assessing energy dependent s t a t e s h i f t s i n i n t a c t leaves. Such s t a t e s h i f t s play an i m p o r t ant r o l e i n the r e g u l a t i o n of p h o t o s y n t h e s i s . ACKNOWLEDGEMENTS We wish to thank U l r i c h Heber f o r s t i m u l a t i n g d i s c u s s i o n s . Support by the Deutsche Forschungsgemeinschaft i s g r a t e f u l l y acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
A l l e n JF and Bennett J (1983) FEBS L e t t 1 2 3 : 6 7 - 7 0 Andersson B and Anderson JM (1980) Biochim Biophys Acta 5 9 3 : 4 2 7 - 4 4 0 Anderson JM (1981) FEBS L e t t 1 2 4 : 1 - 1 0 Barber J and Chow WS (1979) FEBS L e t t 1 0 5 : 5 - 1 0 Bennett J, Steinback KE and Arntzen CJ (1980) Proc Natl Acad Sci USA 77:5253-5257 Bennett J (1983) Biochem J 2 1 2 : 1 - 1 3 Bouges-Boequet B (1984) Biochim Biophys Acta 3 1 4 : 2 5 0 - 2 5 6 Bradbury M and Baker NR (1981) Biochim Biophys Acta 6 3 5 : 5 4 2 - 5 5 1 Bradbury M and Baker NR (1984) Biochim Biophys Acta 7 6 5 : 2 7 5 - 2 8 1 B r i a n t a i s JM, Vernotte C, Picaud M and Krause GH (1980) Biochim Biophys Acta 5 9 1 : 1 9 8 - 2 0 2 B r i a n t a i s JM, Vernotte C, Krause GH and Weis E (1986) In L i g h t Emission by Plants and B a c t e r i a (Govindjee, Amesz J and Fork DC, eds.) Academic Press, i n press, New York B u t l e r WL and K i t a j i m a M (1975) Proc 3rd Congr Photosynth, Vol. I , pp. 13-24 B u t l e r WL (1977) In Encyclopedia o f P l a n t Physiology (Trebst A and Avron M eds.) Vol. V, pp. 149-167, Springer Verlag, Heidelberg D i e t z KJ, Schreiber U and Heber U (1985) Planta 1 6 6 : 2 1 9 - 2 2 6 Hodges M and Barber J (1983) Plant P h y s i o l 7 2 : 1 1 1 9 - 1 1 2 2 Horton P and Black MT (1980) FEBS L e t t 1 1 9 : 1 4 1 - 1 4 4 Horton P and Black MT (1981) Biochim Biophys Acta 6 3 5 : 5 3 - 8 2 Horton P, A l l e n JF, Black MT and Bennett J (1981) FEBS L e t t 125: 193-196 Krause GH, B r i a n t a i s JM and Vernotte C (1983) Biochim Biophys Acta 723:169-175 Krause GH, Behrend U (1983) Biochim Biophys Acta 7 2 3 : 1 7 6 - 1 8 1 Krause GH and Weis E (1984) Photosynth Res 5 : 1 3 9 - 1 5 7 Lavorel J and Etienne AL (1977) In Primary Processes o f Photosynthesis (Barber J, ed.) pp. 202-268 E l s e v i e r , Amsterdam
308
[162l
23. Quick P and Horton P (1984) Proc R Soc Lond 2 2 0 : 3 6 1 - 3 7 0 24. Quick P and Horton P (1984) Proc R Soc Lond 2 2 0 : 3 7 1 - 3 8 2 25. Renger G and Schreiber U (1986) In Light Emission by Plants and Bacteria (Govindjee, Amesz d and Pork DC, eds.) Academic Press, in press, New York 26. Schreiber U, Bilger W and Schliwa U (1985) Photosynth 8es, in press 27. Schreiber U (1986) Special Issue of Photosynth Res, in press 28. Strasser RJ and Butler WL (1977) Biochim Biophys Acta 462:295-306 29. Teller A, Bottin H, Barber d and Mathis P (1984) Biochim Biophys Acta 764:324-330 30. T h i e l e n APG and van Gorkom HJ (1981) FEBS L e t t 1 2 9 : 5 7 - 6 1 31. Velthuys BR and Amesz J (1974) Biochim Biophys Acta 3 3 3 : 8 5 - 9 4 Note added a f t e r manuscript submission: Very r e c e n t l y Malkin, T e l l e r and Barber (Biochim. Biophys. Acta 848, 48-. 57, 1986) reported on fluorescence measurements with a conventional modulation technique, suggesting changes in absorption cross-section of the two photosystems accompanying changes in F 0 and F v during Light 1 - Light 2 induced state changes. While our findings do agree with the conclusions of these authors, it should be noted that there is a principal difference in the definition of F 0 in the approach of Malkin and co-workers as compared to our study. These authors determine "Fo" using a measuring light of 7 W/m 2 which is 7000 times stronger than what was applied here, and which is sufficiently intensive to induce appreciable variable fluorescence. Hence, the observed changes in "Fo" could well represent, at least in part, changes in Fv.Even if,as suggested by Malkin (Isr. d. Chem. 21, 306-315, 1981), the F v which can not be quenched by Light 1 originated from "unconnected PS II centers", it remains uncertain to what extent the relative size of this population of centers may vary during state changes. Such variation would cause changes in F v mimicking changes in "Fo". Furthermore, besides of non-photochemical quenching induced by a state 1 - state 2 transition, also changes due to membrane energization (qE) should be considered. Therefore, we believe that for definite demonstration of true changes in absorption cross-section the actual F0 has to be measured.
