Photosynthesis Research 25: 249-257, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.
Regular paper
The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves Bernard Genty ~, Jeremy Harbinson 2, Jean-Marie Briantais 1'3 & Neil R. Baker 1'4
tDepartment of Biology, University of Essex, Colchester, C04 3SQ, Essex, UK; 2AFRC Institute of Plant Science Research, John Innes Institute, Colney Lane, Norwich, NR4 7UH, UK; 3permanent address: Laboratoire d'Ecologie V~g(tale, Bat 362, Universit~ Paris-Sud, 91405 Orsay, Cedex, France; 4To whom all correspondence regarding this manuscript and reprint requests should be addressed Received 11 October 1989; accepted in revised form 14 May 1990
Key words: Chlorophyll fluorescence, flash-induced kinetics, fluorescence quenching, non-photochemical quenching, photochemistry, photosystem 2 Abstract It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that nonphotochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.
Abbreviations: F - fluorescence yield (subscripts o, i, m, s, v, f def'me minimal, i, maximal, steady-state, variable and single turnover flash induced levels, respectively): A F - 1 s saturating pulse-induced fluorescence yield change; AFf - single turnover flash-induced fluorescence yield change; AFemox- single turnover saturating flash-induced fluorescence yield change; P P F D - photosynthetic photon flux density; PQ - plastoquinone; PS 2 - photosystem 2; QA - primary quinone-type electron acceptor of PS 2; q E - energy-dependent quenching of chlorophyll fluorescence; q N - non-photochemical quenching of chlorophyll fluorescence; q p - photochemical quenching of chlorophyll fluorescence
Introduction It is well established that the yield of chlorophyll fluorescence emission from photosynthetic organisms is determined by two distinct processes, photochemical (qp) and non-photochemical (qN) quenching (Bradbury and Baker 1981, Schreiber
et al. 1986). Efforts to quantitate the relationships between these fluorescence quenching processes and electron transport in vivo (Weis and Berry 1987, Horton and Hague 1988) have led to the proposal that a component of non-photochemical quenching associated with energization of the thylakoids (often designated q~) is associ-
250 ated with a thermal deactivation of PS 2 excitation and consequently produces a decrease in the quantum efficiency of PS 2 photochemistry (Weis and Berry 1987). Recently we have demonstrated quantititavely that the increase in qN induced by increasing the light intensity from zero to that required to saturate photosynthesis in leaves, when considered in conjunction with the decrease in the concentration of open (oxidized) PS 2 reaction centres, accounts for the observed decreases in the quantum yield of noncyclic electron transport in the leaves (Genty et al. 1989). Such observations suggest that the non-photochemical processes associated with qN may be mediated in the pigment matrices. In this study the effect of light-induced increases of qN on the photochemical efficiency of open PS 2 reaction centres in leaves is examined. Using an approach introduced by Mauzerall (Mauzerall 1978, Falkowski et al. 1986), analyses of light dosage response curves of the fluorescence yield induced by a single turnover flash in the presence of continuous actinic light, which produces varying levels of steady state electron transport and qN, are used to examine the relationship between qN and the exciton capture efficiency of PS 2 reaction centres. Measurements are also made of the rapid, photochemically-induced rise, in fluorescence (from F 0 to Fq) in the presence and absence of qN, when leaves are exposed to actinic light levels saturating for PS 2 photochemistry (Morin 1964, DeIosme 1967, Neubauer and Schreiber 1987). The data presented supports strongly the contention that the processes in the thylakoids associated with qN can determine the photochemical efficiency of PS 2.
Materials and methods
Mature leaves were obtained from plants of pea (Pisum sativum var BCI/8RR) and barley (Hordeum vulgare var Clermont) which were grown at a mean temperature of 20°C in a glasshouse supplemented with artificial light to give a minimum photosynthetically-active photon flux density (PPFD) of 550/zmolm-Zs -~ for a 16h photoperiod. All leaves were dark-adapted for a
minimum of l h prior to measurements being made. Measurements of the yields of modulated chlorophyll fluorescence emission were made as described previously (Genty et al. 1989) using a pulse amplitude modulation fluorimeter (PAM 101, Walz, Schreiber 1986). For leaves exposed to any given set of excitation conditions the minimal (F0), maximal (Fro) and steady-state (Fs) levels of fluorescence yield were determined at the steady-state of photosynthesis (achieved after at least 30 min). F m was estimated from the fluorescence yield achieved on addition of a 0.5 s pulse of saturating white actinic light having a PPFD of 7500/~mol m -2 s -1. F 0 was measured by the addition of 30 t~molm-2s -~ of far-red (peak wavelength 710nm) light on removal of the actinic light from the leaf. The coefficients for photochemical (qp) and non-photochemical (qN) quenching, the photochemical efficiency of open PS 2 reaction centres (estimated by Fv/Fm) and the quantum efficiency of PS 2 photochemistry (estimated by AF/Fm, where AF= F m - F s ) were determined as described previously (Genty et al. 1989). Measurements of the increase and following decay of the fluorescence yield induced by a single turnover flash were made on leaves concomitantly with determinations of F 0, F m and F S using the pulse amplitude fluorimeter as described above except with minimal damping (low pass filter on the output of recent instruments removed). A xenon flash tube (15J, E.G. and G.) in conjunction with a Schott BG39 glass filter produced a blue-green flash with a half peak width of 3 ~s. The energy of the flash at the leaf surface was attenuated using calibrated neutral density glass filters. Flash-induced fluorescence yield changes were recorded 130/.~s after the flash; this delay was essential to enable reliable measurements to be made after the 90 ~s gating period of the detector during the flash. The kinetics of fluorescence yield changes were captured using a transient recorder (DSA524, Thurlby) and analyzed with a microcomputer. For leaves exposed to actinic light data from a sequence of 16 to 32 flashes with a repetition frequency of 0.5 Hz was averaged. For dark-adapted leaves a repetition rate of 180 s was used to ensure maximal oxidation of QA between
251 each flash (Chylla et al. 1987). The flash-induced changes in fluorescence yield were expressed as AFf (AFf : F f - F~) where Ff is the fluorescence yield induced by a single turnover flash and measured after 170/xs, and F~ is the steady-state fluorescence yield (see above) before the flash. Consequently AFf is defined as the flash-induced fluorescence yield change at 170/zs after the flash. The photochemically-induced rises in fluorescence in saturating light from F 0 to the initial inflection point, F~ as defined by Neubauer and Schreiber (1987), were measured from leaves exposed to a 300 tzs pulse of 660 nm light (PPFD 3 0 0 0 0 / x m o l m - 2 s -1) produced from 36 red light-emitting diodes (Stanley H3000) focused onto a 1 cm leaf area. The light-emitting diodes were driven by a custom-built power supply capable of switching the light on and off in 500 ns. Fluorescence was detected with a BPX65 pin photodiode protected by a near infra-red longpass filter (Kodak Wratten 88A). Signals were preamplified with an operational amplifier (Burr Brown OPA606) with a frequency response of 1 MHz and recorded with the transient recorder described above.
Results and discussion
Following a saturating, single turnover flash fluorescence yield has been shown to remain relatively constant at its maximum value over a time range of 10 to 100/zs (Crofts and Wraight 1983). During this time domain quenchers of fluoresence are absent; the short-lived products of reaction centre photochemistry, e.g., P680 ÷, which can quench fluorescence, will have decayed, and QA, the primary quinone electron acceptor of PS2, remains reduced (Mauzerall 1972). After 100/xs, the flash-induced fluorescence yield declines to the pre-flash value. The kinetics of this decay have been shown to be complex and follow the oxidation of QA by the secondary quinone electron acceptor, QB, via a two electron gate (Crofts and Wraight 1983, Zankel 1973, Bouges Bouquet 1973, Velthuys and Amesz 1974). Figure 1 shows the fluorescence yield decay kinetics from a dark-adapted pea leaf at times greater than 130/xs after a saturating
~
T
o o
20Op~
T flash
time --.
Fig. 1. The kinetics of the decay of fluorescence yield measured 130/.ts after a dark-adapted pea leaf is given a saturating, single turnover flash (half-peak width 3 p.s).
single turnover flash given with a frequency of 5.5 × 10 -3 Hz. For a dark-adapted leaf exposed to this flash repetition rate (one flash given every 180 s), it is expected that the dark equilibrium between QA and QB will be achieved between each flash (Chylla et al. 1987). Under these conditions the decay of fluorescence yield has a half-time of c a . 400 ~s and can be deconvoluted into two major exponential components with rate constants (k) and relative amplitudes (A) of ca. k l = 3 × 1 0 3 s -1, A 1 = 0 . 7 5 and k 2 = 5 × 102 s -1, A 2 = 0.25, respectively. A third, slower component of small amplitude (A < 0.12) in the very slow time domain of 100 ms-10 s was not taken into account in this deconvolution analysis (Chylla et al. 1987). This complex fluorescence decay is similar to those observed previously in isolated chloroplasts and algae (Crofts and Wraight 1983, Robinson and Crofts 1983) and reflects the two electron gate mechanism for electron transfer from QA to QB. On exposure of leaves to increasing levels of continuous actinic light, an increasing fraction of PS 2 reaction centres become reduced (closed) at steady-state photosynthesis, as indicated by the decrease in qp with light intensity (Table 1). Closure of PS 2 reaction centres is also accompanied by large increases in qN with increasing light intensity (Table 1). Light intensity-induced increases in qN correspond with decreases in Fv/F m (Table 1), which indicate a decrease in the photochemical efficiency of open (oxidized) PS 2 reaction centres (Genty et al. 1989). The decreases in qp and Fv/F m with increasing actinic light intensity result in a decrease in the quantum
252 efficiency of PS 2 photochemistry, ~PS 2' (Table 1), which is determined by the product of qp and Fv/F m ( G e n t y et al. 1989). W h e n leaves exposed to different intensities of actinic light were given a saturating, single turnover flash, a decrease in the flash-induced fluorescence yield (measured 170/zs after the flash), Ff, was observed with increasing irradiance (Table 1). H o w e v e r , when the magnitude of the difference (AFf) between Fy and the steady-state fluorescence yield before the flash, F s, (i.e., Ff = F y - Fs) , was normalized to give equal values at all continuous irradiances to which the leaf was exposed, no significant differences were observed between the decay kinetics of the flash-induced fluorescence yield (data not shown) as has been shown previously in algae (Falkowski et al. 1986). Consequently the rate at which AFf relaxes to the steady-state fluorescence level in the actinic, continuous light (Fs), following the saturating, single turnover flash, is independent of the intensity of the actinic light even though both qv and qN have changed dramatically (Table 1). These data indicate that the rate of reoxidation of QA after a flash is independent of both the fraction of open P S 2 reaction centres and the degree of energization of the thylakoids. The qN induced decrease in A~'f (proclucea oy a single turnover flash) is similar to that induced by qN in AF (produced by a 1 s light pulse) when measured over the range of actinic P P F D 4 5 1582/zmol m -2 s -1 (Table 1, Fig. 2). H o w e v e r , m e a s u r e m e n t of d a r k - a d a p t e d leaves c o m p a r e d to leaves exposed to a P P F D of 4 5 / x m o l m -2 s -1 d e m o n s t r a t e d a decrease of AF in the absence of any change in AFf; this could be interpreted as a change in the a m o u n t of plastoquinone quenching at F s due to the reduction of plastoquinone in the light (Vernotte et al. 1979). It has been
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Regular paper
The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves Bernard Genty ~, Jeremy Harbinson 2, Jean-Marie Briantais 1'3 & Neil R. Baker 1'4
tDepartment of Biology, University of Essex, Colchester, C04 3SQ, Essex, UK; 2AFRC Institute of Plant Science Research, John Innes Institute, Colney Lane, Norwich, NR4 7UH, UK; 3permanent address: Laboratoire d'Ecologie V~g(tale, Bat 362, Universit~ Paris-Sud, 91405 Orsay, Cedex, France; 4To whom all correspondence regarding this manuscript and reprint requests should be addressed Received 11 October 1989; accepted in revised form 14 May 1990
Key words: Chlorophyll fluorescence, flash-induced kinetics, fluorescence quenching, non-photochemical quenching, photochemistry, photosystem 2 Abstract It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that nonphotochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.
Abbreviations: F - fluorescence yield (subscripts o, i, m, s, v, f def'me minimal, i, maximal, steady-state, variable and single turnover flash induced levels, respectively): A F - 1 s saturating pulse-induced fluorescence yield change; AFf - single turnover flash-induced fluorescence yield change; AFemox- single turnover saturating flash-induced fluorescence yield change; P P F D - photosynthetic photon flux density; PQ - plastoquinone; PS 2 - photosystem 2; QA - primary quinone-type electron acceptor of PS 2; q E - energy-dependent quenching of chlorophyll fluorescence; q N - non-photochemical quenching of chlorophyll fluorescence; q p - photochemical quenching of chlorophyll fluorescence
Introduction It is well established that the yield of chlorophyll fluorescence emission from photosynthetic organisms is determined by two distinct processes, photochemical (qp) and non-photochemical (qN) quenching (Bradbury and Baker 1981, Schreiber
et al. 1986). Efforts to quantitate the relationships between these fluorescence quenching processes and electron transport in vivo (Weis and Berry 1987, Horton and Hague 1988) have led to the proposal that a component of non-photochemical quenching associated with energization of the thylakoids (often designated q~) is associ-
250 ated with a thermal deactivation of PS 2 excitation and consequently produces a decrease in the quantum efficiency of PS 2 photochemistry (Weis and Berry 1987). Recently we have demonstrated quantititavely that the increase in qN induced by increasing the light intensity from zero to that required to saturate photosynthesis in leaves, when considered in conjunction with the decrease in the concentration of open (oxidized) PS 2 reaction centres, accounts for the observed decreases in the quantum yield of noncyclic electron transport in the leaves (Genty et al. 1989). Such observations suggest that the non-photochemical processes associated with qN may be mediated in the pigment matrices. In this study the effect of light-induced increases of qN on the photochemical efficiency of open PS 2 reaction centres in leaves is examined. Using an approach introduced by Mauzerall (Mauzerall 1978, Falkowski et al. 1986), analyses of light dosage response curves of the fluorescence yield induced by a single turnover flash in the presence of continuous actinic light, which produces varying levels of steady state electron transport and qN, are used to examine the relationship between qN and the exciton capture efficiency of PS 2 reaction centres. Measurements are also made of the rapid, photochemically-induced rise, in fluorescence (from F 0 to Fq) in the presence and absence of qN, when leaves are exposed to actinic light levels saturating for PS 2 photochemistry (Morin 1964, DeIosme 1967, Neubauer and Schreiber 1987). The data presented supports strongly the contention that the processes in the thylakoids associated with qN can determine the photochemical efficiency of PS 2.
Materials and methods
Mature leaves were obtained from plants of pea (Pisum sativum var BCI/8RR) and barley (Hordeum vulgare var Clermont) which were grown at a mean temperature of 20°C in a glasshouse supplemented with artificial light to give a minimum photosynthetically-active photon flux density (PPFD) of 550/zmolm-Zs -~ for a 16h photoperiod. All leaves were dark-adapted for a
minimum of l h prior to measurements being made. Measurements of the yields of modulated chlorophyll fluorescence emission were made as described previously (Genty et al. 1989) using a pulse amplitude modulation fluorimeter (PAM 101, Walz, Schreiber 1986). For leaves exposed to any given set of excitation conditions the minimal (F0), maximal (Fro) and steady-state (Fs) levels of fluorescence yield were determined at the steady-state of photosynthesis (achieved after at least 30 min). F m was estimated from the fluorescence yield achieved on addition of a 0.5 s pulse of saturating white actinic light having a PPFD of 7500/~mol m -2 s -1. F 0 was measured by the addition of 30 t~molm-2s -~ of far-red (peak wavelength 710nm) light on removal of the actinic light from the leaf. The coefficients for photochemical (qp) and non-photochemical (qN) quenching, the photochemical efficiency of open PS 2 reaction centres (estimated by Fv/Fm) and the quantum efficiency of PS 2 photochemistry (estimated by AF/Fm, where AF= F m - F s ) were determined as described previously (Genty et al. 1989). Measurements of the increase and following decay of the fluorescence yield induced by a single turnover flash were made on leaves concomitantly with determinations of F 0, F m and F S using the pulse amplitude fluorimeter as described above except with minimal damping (low pass filter on the output of recent instruments removed). A xenon flash tube (15J, E.G. and G.) in conjunction with a Schott BG39 glass filter produced a blue-green flash with a half peak width of 3 ~s. The energy of the flash at the leaf surface was attenuated using calibrated neutral density glass filters. Flash-induced fluorescence yield changes were recorded 130/.~s after the flash; this delay was essential to enable reliable measurements to be made after the 90 ~s gating period of the detector during the flash. The kinetics of fluorescence yield changes were captured using a transient recorder (DSA524, Thurlby) and analyzed with a microcomputer. For leaves exposed to actinic light data from a sequence of 16 to 32 flashes with a repetition frequency of 0.5 Hz was averaged. For dark-adapted leaves a repetition rate of 180 s was used to ensure maximal oxidation of QA between
251 each flash (Chylla et al. 1987). The flash-induced changes in fluorescence yield were expressed as AFf (AFf : F f - F~) where Ff is the fluorescence yield induced by a single turnover flash and measured after 170/xs, and F~ is the steady-state fluorescence yield (see above) before the flash. Consequently AFf is defined as the flash-induced fluorescence yield change at 170/zs after the flash. The photochemically-induced rises in fluorescence in saturating light from F 0 to the initial inflection point, F~ as defined by Neubauer and Schreiber (1987), were measured from leaves exposed to a 300 tzs pulse of 660 nm light (PPFD 3 0 0 0 0 / x m o l m - 2 s -1) produced from 36 red light-emitting diodes (Stanley H3000) focused onto a 1 cm leaf area. The light-emitting diodes were driven by a custom-built power supply capable of switching the light on and off in 500 ns. Fluorescence was detected with a BPX65 pin photodiode protected by a near infra-red longpass filter (Kodak Wratten 88A). Signals were preamplified with an operational amplifier (Burr Brown OPA606) with a frequency response of 1 MHz and recorded with the transient recorder described above.
Results and discussion
Following a saturating, single turnover flash fluorescence yield has been shown to remain relatively constant at its maximum value over a time range of 10 to 100/zs (Crofts and Wraight 1983). During this time domain quenchers of fluoresence are absent; the short-lived products of reaction centre photochemistry, e.g., P680 ÷, which can quench fluorescence, will have decayed, and QA, the primary quinone electron acceptor of PS2, remains reduced (Mauzerall 1972). After 100/xs, the flash-induced fluorescence yield declines to the pre-flash value. The kinetics of this decay have been shown to be complex and follow the oxidation of QA by the secondary quinone electron acceptor, QB, via a two electron gate (Crofts and Wraight 1983, Zankel 1973, Bouges Bouquet 1973, Velthuys and Amesz 1974). Figure 1 shows the fluorescence yield decay kinetics from a dark-adapted pea leaf at times greater than 130/xs after a saturating
~
T
o o
20Op~
T flash
time --.
Fig. 1. The kinetics of the decay of fluorescence yield measured 130/.ts after a dark-adapted pea leaf is given a saturating, single turnover flash (half-peak width 3 p.s).
single turnover flash given with a frequency of 5.5 × 10 -3 Hz. For a dark-adapted leaf exposed to this flash repetition rate (one flash given every 180 s), it is expected that the dark equilibrium between QA and QB will be achieved between each flash (Chylla et al. 1987). Under these conditions the decay of fluorescence yield has a half-time of c a . 400 ~s and can be deconvoluted into two major exponential components with rate constants (k) and relative amplitudes (A) of ca. k l = 3 × 1 0 3 s -1, A 1 = 0 . 7 5 and k 2 = 5 × 102 s -1, A 2 = 0.25, respectively. A third, slower component of small amplitude (A < 0.12) in the very slow time domain of 100 ms-10 s was not taken into account in this deconvolution analysis (Chylla et al. 1987). This complex fluorescence decay is similar to those observed previously in isolated chloroplasts and algae (Crofts and Wraight 1983, Robinson and Crofts 1983) and reflects the two electron gate mechanism for electron transfer from QA to QB. On exposure of leaves to increasing levels of continuous actinic light, an increasing fraction of PS 2 reaction centres become reduced (closed) at steady-state photosynthesis, as indicated by the decrease in qp with light intensity (Table 1). Closure of PS 2 reaction centres is also accompanied by large increases in qN with increasing light intensity (Table 1). Light intensity-induced increases in qN correspond with decreases in Fv/F m (Table 1), which indicate a decrease in the photochemical efficiency of open (oxidized) PS 2 reaction centres (Genty et al. 1989). The decreases in qp and Fv/F m with increasing actinic light intensity result in a decrease in the quantum
252 efficiency of PS 2 photochemistry, ~PS 2' (Table 1), which is determined by the product of qp and Fv/F m ( G e n t y et al. 1989). W h e n leaves exposed to different intensities of actinic light were given a saturating, single turnover flash, a decrease in the flash-induced fluorescence yield (measured 170/zs after the flash), Ff, was observed with increasing irradiance (Table 1). H o w e v e r , when the magnitude of the difference (AFf) between Fy and the steady-state fluorescence yield before the flash, F s, (i.e., Ff = F y - Fs) , was normalized to give equal values at all continuous irradiances to which the leaf was exposed, no significant differences were observed between the decay kinetics of the flash-induced fluorescence yield (data not shown) as has been shown previously in algae (Falkowski et al. 1986). Consequently the rate at which AFf relaxes to the steady-state fluorescence level in the actinic, continuous light (Fs), following the saturating, single turnover flash, is independent of the intensity of the actinic light even though both qv and qN have changed dramatically (Table 1). These data indicate that the rate of reoxidation of QA after a flash is independent of both the fraction of open P S 2 reaction centres and the degree of energization of the thylakoids. The qN induced decrease in A~'f (proclucea oy a single turnover flash) is similar to that induced by qN in AF (produced by a 1 s light pulse) when measured over the range of actinic P P F D 4 5 1582/zmol m -2 s -1 (Table 1, Fig. 2). H o w e v e r , m e a s u r e m e n t of d a r k - a d a p t e d leaves c o m p a r e d to leaves exposed to a P P F D of 4 5 / x m o l m -2 s -1 d e m o n s t r a t e d a decrease of AF in the absence of any change in AFf; this could be interpreted as a change in the a m o u n t of plastoquinone quenching at F s due to the reduction of plastoquinone in the light (Vernotte et al. 1979). It has been
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