Fluorescence quenching analysis

Transcription

1 Fluorescence quenching analysis Author: Jun Minagawa Affiliation: Institute of Low Temperature Science, Hokkaido University, N19 W8, Sapporo , Japan Name and address for correspondence: Jun Minagawa Institute of Low Temperature Science Hokkaido University N19 W8, Sapporo Japan TEL: +81-(0) FAX: +81-(0)

2 A. Introduction Chlorophyll a fluorescence is a highly versatile tool, not only for researchers studying photosynthesis, but also for those working in broader fields related to physiology of plants and green algae. Chlorophyll fluorescence analysis is sensitive, real-time, non-invasive, and relatively simple, but indirect. The section here is meant to guide proper interpretation and familiarize the reader with terminology (for further reading see Maxwell and Johnson, 2000; Falkowski and Raven, 2007). Chlorophyll fluorescence analysis is applicable to chapters 15, 16 and 22 in The Chlamydomonas Sourcebook, Volume 2. Absorption of a photon raises a chlorophyll a molecule to its lowest singlet excited state, for which three internal decay pathways exist: fluorescence, in which the molecule returns to the ground state with the emission of radiation; internal conversion, in which the energy of the molecule is converted into vibrational energy; and intersystem crossing, in which the singlet state is converted to the triplet state. If certain other molecules are present along with the chlorophyll, external decay pathway(s) may also become available in addition to the internal decay pathways. Such external pathways facilitate the transfer of energy to a molecule with a similar energy gap or the transfer of an electron to or from another molecule, such as in excitation energy transfer in light-harvesting antennae and charge separation in photochemical reaction centers, respectively. All of these downward processes competitively contribute to the decay of the chlorophyll excited state. Accordingly, an increase in the rate of one of these processes would increase its share of the decay process and lower the fluorescence yield (φ f ). The quantum yield of chlorophyll fluorescence from the photosynthetic apparatus is therefore 0.6-3%, while chlorophyll a in an organic solvent exhibits a high fluorescence yield of approximately 30% (Latimer et al., 1956; Trissl et al., 1993). Historically, the term quenching refers to all processes that lower φ f. 2

3 B. Quenching analysis Classic experiments showing the relationship between photosynthetic reactions and φ f were made by Kautsky and Hirsh (1931). They recorded a rapid rise of fluorescence from a leaf through colored glass at the onset of illumination, which was followed by a decline to reach a steady level (Kautsky effect). Duysens and Sweers (1963) proposed that changes in φ f are primarily related to the redox state of the electron-transferring components of PS II, and introduced the term Q as a quencher. Butler and Kitajima (1975) elaborated that with Q in the ground state, charge separation can occur and the reaction center is described as being open. When Q is reduced, further stable charge separation is inhibited and the center is described as being closed. Subsequent experiments have shown that Q is identical to Q A in the PS II reaction center. Photosynthetic reactions occur in PS II over a broad time range. Reactions lasting up to hundreds of picoseconds are considered to be ultrafast reactions; these include excitation energy transfer and charge separation and stabilization, which have been described by the exciton-radical pair equilibrium model or its derivatives (Schatz et al., 1988; Laible et al., 1994; van Grondelle et al., 1994). The PS II acceptor-side reactions that include electron sharing between the primary and secondary quinones of PS II (Q A and Q B respectively), the subsequent reactions that result in the reduction of the intersystem electron carriers, and the PS II donorside reactions that include electron donation from the oxygen-evolving complex to the secondary electron donor in PS II (Yz) and from Yz to P680 +, are considered to be the fast reactions; these last from tens of microseconds to tens of milliseconds. Lastly, energydependent q E -quenching, state transitions, and photoinhibition are considered to be the slow more acclimatory reactions; the duration of these reactions ranges from a couple of seconds to hundreds of minutes. Different and specialized techniques have been applied for elucidating the 3

4 reactions in different time domains. Only a brief summary of fluorescence quenching analysis is provided here, which targets the slow reactions. C. Photochemical quenching If all antenna chlorophylls for PS II are similar in their energy levels, the probability of excitation decay is independent of the individual pigment. In a simple scheme, the quantum yield of fluorescence (φ f ) and photochemistry (φ II ) are therefore given by the rate constants for fluorescence (k f ) and photochemistry (k p ), respectively, divided by the sum of the rate constants for all competing processes of de-excitation:!! f II = k f = k f k + k k f p + k p p + k h + k h where k h denotes the rate constant for the thermal dissipation. When Q A is in its oxidized state, the charge stabilization reaction (P680 * PheoQ A P680 + Pheo Q A P680 + PheoQ A ) occurs within hundreds of picoseconds (Renger et al., 1995), which primarily results in the loss of the exciton-radical pair equilibrium state. Thus, the pathway of photochemistry in the open PS II reaction center results in a decrease of φ f, which is termed photochemical quenching. When Q A is in its reduced state, i.e. when the centers are closed, the charge separated state (P680 + Pheo) cannot be stabilized and the value of k p is reduced to zero. The exciton-radical pair equilibrium state then continues where the loss of the excited state would primarily be due to fluorescence. The two levels of fluorescence are defined as follows: Fo (unquenched minimal yield of fluorescence ) is the level of chlorophyll fluorescence when all PS II centers are open and Fm (unquenched maximal yield of fluorescence) is the maximal level of chlorophyll fluorescence when all PS II centers are closed. Practically, Fo and Fm are measured as fluorescence emitted from dark-adapted samples when probed with a weak pulse of measuring light before and after 4

5 a strong saturating flash is given, respectively. The difference between Fm and Fo is defined as Fv (unquenched variable fluorescence). D. Non-photochemical quenching Overexcitation of chlorophyll and overreduction of the electron transport chain can result in increased generation of reactive intermediates and harmful byproducts of photosynthesis. In order to maintain a short lifetime of an excited chlorophyll molecule and protect the organism from damage by excess light absorption, there exist additional decay pathways for de-excitation of chlorophyll molecules in competition with photochemistry, fluorescence, and other decay processes. Fluorescence quenching processes observed under such conditions are collectively called non-photochemical quenching and have been described for a long time (Govindjee et al., 1967; Bonaventura and Myers, 1969; Murata, 1969; Murata and Sugahara, 1969). Classically, mechanisms for inducing non-photochemical quenching have been categorized into 3 classes; 1) high-energy dependent q E -quenching, 2) state transitiondependent q T -quenching, and 3) photoinhibition-dependent q I -quenching (Krause and Weis, 1991). All these processes have been postulated to have physiological roles that contribute to the protection of the photosynthetic apparatus. By comparing fluorescence quenching in samples with or without the suppression of photochemistry, it is possible to distinguish whether it is based on a change in k p or k h, assuming that the intrinsic rate constant for fluorescence (k f ) is not variable. E. Pulsed-amplitude modulation (PAM) fluorometry In order to distinguish between the photochemical and non-photochemical contributions to quenching, the so-called light doubling or saturation pulse method was introduced (Bradbury and Baker, 1981). This technique is based on the assumption that all PS II reaction centers can be transiently closed, thus eliminating the contribution of all photochemical quenching without causing any change in non-photochemical quenching. An innovative device in the application of chlorophyll fluorescence uses a modulated measuring beam (Quick and 5

6 Horton, 1984), where the fluorescence signals are isolated by a detector with a lock-in amplifier, making the accurate separation of signals in the presence of a background light possible. Based on these ideas, pulsed amplitude modulation (PAM) fluorometry was designed (Schreiber et al., 1986). Using this system, fluorescence yield can be measured even in the presence of white background illumination and, most significantly, in the presence of sunlight. In order to close all PS II reaction centers transiently, a high-intensity, short-duration flash of light that lasts for approximately seconds is given. During this saturation pulse, the fluorescence yield reaches a value equivalent to that which would be obtained in the absence of any photochemical quenching. Of particular importance is that Fm (quenched maximal yield of fluorescence) and Fo (quenched minimal yield of fluorescence) can be readily measured in addition to that of the dark-adapted state, Fm and Fo. F. Video imaging analysis The versatility of chlorophyll fluorometry increased significantly with the development of techniques that take advantage of CCD (Charge-Coupled Device) cameras and computerized data processing (Omasa et al., 1987; Fenton and Crofts, 1990; Nedbal et al., 2000; Oxborough, 2004). The typical instrument is illustrated in Fig. 1, with which spatial and temporal changes of various fluorescence parameters can be readily obtained. This has facilitated screens for photosynthetic mutants of various organisms including Chlamydomonas (Bennoun and Béal, 1997; Niyogi et al., 1997; Fleischmann et al., 1999; Kruse et al., 1999), and allowed comparisons of photosynthetic performance on different leaf spots (Daley et al., 1989; Siebke and Weis, 1995). 6

7 7

8 G. Parameters used in quenching analysis Calculation of fluorescence parameters is best explained by referring to a typical experimental trace obtained with a PAM-type fluorometer (Fig. 2). Measurement is initiated by switching on the weak measuring light, providing a measure of Fo (upward arrow). A saturating flash of light or multiple subsaturating flashlets that together trigger single turnover excitation (filled arrowheads), is then applied and allows the measurement of Fm from the dark-adapted sample. Next, actinic light (open bar) is applied and further saturating pulses are given at appropriate intervals to measure Fm. The transient fluorescence, Ft, is monitored during the entire duration. The fluorescence minimum in the quenched state, Fo, is measured immediately after the removal of actinic light (downward arrow). The variable fluorescence in the dark and in the quenched state (Fv ) is expressed by Fv 8

9 = Fm Fo, and Fv = Fm Fo, respectively. Resolution of quenching parameters provides a variety of information on the functional state of the photosynthetic apparatus as described below. These parameters are the ones used most often in the literature. 1. Fo Fo is the minimal yield of fluorescence from samples in the quenched state and is usually measured immediately after the removal of actinic light, so that the fluorescence reflects the nature of the samples in the actinic light at a time when all Q A is oxidized. By comparing Fo with Fo, one can estimate if the fluorescence quenching observed in Fm is due to quenching in a portion of Fv or Fo. Measurement of this parameter is sometimes problematic, as a large proportion of non-photochemical quenching quickly follows changes in the ΔpH, which also decays rapidly once the illumination is removed. To quantify this potential source of artifacts, Oxborough and Baker (1997) estimated Fo through a simple equation involving only Fo, Fm, and Fm. The formalization is as follows: minimal fluorescence (quenched) : Fo! = Fo Fv Fo + Fm Fm! They demonstrated that the calculated value of Fo correlates well with the experimentally measured Fo (Oxborough and Baker, 1997). 2. Quantum yield of photochemistry (Fv / Fm, Fv / Fm, ΔF / Fm, φ II ) Probably the most useful fluorescence parameter measures the proportion of absorbed energy used in photochemistry, namely the quantum yield of photochemistry. If measured in the dark, the intrinsic quantum yield is expressed as Fv / Fm (Kitajima and Butler, 1975). In the presence of light, the intrinsic and effective quantum yields are calculated as Fv / Fm and ΔF / Fm, respectively (Genty et al., 1989). φ II has been used for quantum yield of photochemistry in general, but, in a more limited sense, it is used for effective quantum yield in the light. 9

10 intrinsic quantum yield after dark adaptation : Fv Fm! Fo = Fm Fm intrinsic quantum yield during illumination : effective quantum yield during illumination : Fv! Fm! " Fo! = Fm! Fm! " F Fm! # Ft $ II = = Fm! Fm! The effective quantum yield in the light (φ II ) is correlated with the quantum yield of CO 2 assimilation under laboratory conditions, where linear electron flow is dominant (Fryer et al., 1998). This parameter is the easiest one to measure; a measurement can be performed by simply applying a fluorometer to a sample and measuring fluorescence before and after a saturating pulse (Ft, Fm ). The dark-adapted values of Fv / Fm are sensitive indicators of a maximal photosynthetic performance, with optimal values of around measured for most plant species (Björkman and Demmig, 1987). Sustained heat dissipation with a low Fv / Fm is often seen when a sample has been exposed to photoinhibitory conditions. If photochemical efficiency of a plant was temporarily suppressed due to reversible non-photochemical quenching, the intrinsic quantum yield would be decreased only in the light, e.g. Fv / Fm would be lowered while Fv / Fm would remain the same. 3. Proportion of open PS II centers (q P, q L ) Another widely used fluorescence parameter is photochemical quenching, q P. This is calculated as: proportion of open PS II centers : q P Fm! " Ft # F = = Fm! " Fo! Fv! While φ II is the proportion of absorbed energy used in photochemistry, q P indicates the proportion of open PS II centers. The alternative expression, 1- q P, reflects the proportion of closed centers, which is sometimes termed the excitation pressure on PS II (Maxwell et al., 1994). proportion of closed PS II centers (excitation pressure) : 1- q P 10

11 q P and φ II can be interrelated by Fv / Fm (Oxborough and Baker, 1997):! F " II = Fm#! F Fv# = $ Fv# Fm# Fv# = qp $ Fm# It is of note that while φ II is related to the overall efficiency of photochemistry, q P and Fv / Fm provide information about the underlying processes. A decrease in q P is due to an increase in the proportion of closed PS II centers, which could be brought about by any defects in the components downstream of PS II, such as inhibition in PS I. A decrease in Fv / Fm, on the other hand, can be caused by any defect in the quantum yield of PS II itself, such as nonphotochemical quenching. Either case could decrease φ II. The parameter q P is based on the so-called puddle antenna model, where each PS II center possesses its own independent antenna system. However, accumulating evidence has suggested that the real photosynthetic units are connected by shared antennae, the lake antenna. Therefore, Kramer et al. (2004) extended the approach by Joliot and Joliot (1964), who had described the non-linear relationship of concentration of Q - A and variable fluorescence, and derived a parameter q L to represent a realistic estimate of the fraction of open PS II centers with a high connectivity of PS II units. proportion of open PS II centers (corrected) : q L Fm! " Ft Fo! = # Fm! " Fo! Ft Fo! = qp # Ft 4. Electron transfer rate (ETR) Since φ II is the quantum yield of PS II photochemistry, it can be used to calculate the rate of linear electron transfer (ETR; Genty et al., 1989): electron transfer rate : ETR =! II " PFDa "

12 where PFDa is absorbed light (µmol photon m -2 s -1 ) and 0.5 is an assumed factor that accounts for the partitioning of energy between PS II and PS I. This assumption could be reasonable during steady state when the PS I and PS II turnover rates are equal. Care should be taken when one encounters conditions where cyclic electron transfer around PS I or PS II is predominant. 5. Non-photochemical quenching (NPQ, qn) The study of energy dissipation processes has been complicated by the fact that researchers have used different terms for non-photochemical quenching, e.g. the parameters NPQ vs qn. The most straightforward way of quantifying non-photochemical quenching is by measuring the quenched fraction of maximal fluorescence, namely the Stern-Volmer-type equation (Bilger and Björkman, 1990): non-photochemical quenching : Fm " Fm! NPQ = Fm! NPQ is linearly related to heat dissipation and lies on a scale from zero to infinity. In a typical photosynthetic organism, values might be expected in the range at saturating light intensities; however, this varies markedly between species and acclimatization conditions. When grown under normal light and temperature conditions, Chlamydomonas exhibits a low value of NPQ (Finazzi et al., 2006). Another term for quantifying non-photochemical quenching is q N (van Kooten and Snel, 1990): non-photochemical quenching : q N Fm " Fm! = Fm " Fo This parameter requires measurement of Fo and falls on a scale of 0-1 and is therefore relatively insensitive to changes in quenching at higher values. 12

13 References Bennoun, P., and Béal, D. (1997). Screening algal mutant colonies with altered thylakoid electrochemical gradient through fluorescence and delayed luminescence digital imaging. Photosynth. Res. 51, Bilger, W., and Björkman, O. (1990). Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth. Res. 25, Björkman, O., and Demmig, B. (1987). Photon yield of O 2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, Bonaventura, C., and Myers, J. (1969). Fluorescence and oxygen evolution from Chlorella pyrenoidosa. Biochim. Biophys. Acta 189, Bradbury, M., and Baker, N. R. (1981). Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of photosystem II electron acceptors and fluorescence emission from photosystems I and II. Biochim. Biophys. Acta 635, Butler, W. L., and Kitajima, M. (1975). Fluorescence quenching in photosystem II of chloroplasts. Biochim. Biophys. Acta 376, Daley, P. F., Raschke, K., Ball, J. T., and Berry, J. A. (1989). Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence. Plant Physiol. 90, Duysens, L. N. M., and Sweers, H. E. (1963). Mechanism of two photochemical reactions in algae as studied by means of luorescence. In "Studies on Microalgae and Photosynthetic Bacteria" (Japanese Society of Plant Physiologists, Ed.), pp University of Tokyo Press, Tokyo. Falkowski, P. G., and Raven, J. A. (2007). Aquatic Photosynthesis: Second Edition. Princeton University Press, Princeton. Fenton, J., M., and Crofts, A., R. (1990). Computer aided fluorescence imaging of photosynthetic systems. Photosynth. Res. 26, Finazzi, G., Johnson, G. N., Dall'Osto, L., Zito, F., Bonente, G., Bassi, R., and Wollman, F.-A. (2006). Nonphotochemical quenching of chlorophyll fluorescence in Chlamydomonas reinhardtii. Biochemistry 45, Fleischmann, M. M., Ravanel, S., Delosme, R., Olive, J., Zito, F., Wollman, F.-A., and Rochaix, J.-D. (1999). Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition. J. Biol. Chem. 274, Fryer, M. J., Andrews, J. R., Oxborough, K., Blowers, D. A., and Baker, N. R. (1998). Relationship between CO 2 assimilation, photosynthetic electron transport, and active O 2 metabolism in leaves of maize in the field during periods of low temperature. Plant Physiol. 116, Genty, B., Briantais, J.-M., and Baker, N. R. (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, Govindjee, Papageorgiou, G., and Rabinowitch, E. (1967). In "Fluorescence; Theory, Instrumentation and Practice" (G. G. Guilbault, Ed.), pp Marcel Dekker, New York. Joliot, A., and Joliot, P. (1964). Étude cinétique de la réaction photochimique libérant l'oxygène au cours de la photosynthèse. C. R.. Acad. Sci. Paris 258, Kautsky, H., and Hirsch, A. (1931). Kurze Originalmitteilungen. Naturwissenschaften 19,

14 Kitajima, M., and Butler, W. L. (1975). Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim. Biophys. Acta 376, Kramer, D. M., Johnson, G., Kiirats, O., and Edwards, G. E. (2004). New fluorescence parameters for the determination of Q A redox state and excitation energy fluxes. Photosynth. Res. 79, Krause, G. H., and Weis, E. (1991). Chlorophyll fluorescence and photosynthesis: The basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, Kruse, O., Nixon, P. J., Schmid, G. H., and Mullineaux, C. W. (1999). Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging. Photosynth. Res. 61, Laible, P. D., Zipfel, W., and Owens, T. G. (1994). Excited state dynamics in chlorophyll-based antennae: the role of transfer equilibrium. Biophys. J. 66, Latimer, P., Bannister, T. T., and Rabinowitch, E. (1956). Quantum yields of fluorescence of plant pigments Science 124, Maxwell, D. P., Falk, S., Trick, C. G., and Huner, N. (1994). Growth at low temperature mimics high-light acclimation in Chlorella vulgaris. Plant Physiol. 105, Maxwell, K., and Johnson, G. N. (2000). Chlorophyll fluorescence - a practical guide. J. Exp. Bot. 51, Murata, N. (1969). Control of excitation transfer in photosynthesis I. Light-induced change of chlorophyll a fluoresence in Porphyridium cruentum. Biochim. Biophys. Acta 172, Murata, N., and Sugahara, K. (1969). Control of excitation transfer in photosynthesis. III. Lightinduced decrease of chlorophyll a fluorescence related to photophosphorylation system in spinach chloroplasts. Biochim. Biophys. Acta 189, Nedbal, L., Soukupova, J., Kaftan, D., Whitmarsh, J., and Trtílek, M. (2000). Kinetic imaging of chlorophyll fluorescence using modulated light. Photosynth. Res. 66, Niyogi, K. K., Björkman, O., and Grossman, A. R. (1997). Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching. Plant Cell 9, Omasa, K., Shimazaki, K. I., Aiga, I., Larcher, W., and Onoe, M. (1987). Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves. Plant Physiol. 84, Oxborough, K. (2004). Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance. J. Exp. Bot. 55, Oxborough, K., and Baker, N. R. (1997). Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components: calculation of qp and Fv'/Fm' without measuring Fo'. Photosynth. Res. 54, Quick, W. P., and Horton, P. (1984). Studies on the induction of chlorophyll fuorescence in barley protoplasts. II. Resolution of fluorescence quenching by redox state and the transthylakoid ph gradient. Philos. Trans. R. Soc. Lond. B Biol. Sci. 220, Schatz, G. H., Brock, H., and Holzwarth, A. R. (1988). Kinetic and energetic model for the primary processes in photosystem II. Biophys. J. 54, Schreiber, U., Schliwa, U., and Bilger, W. (1986). Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10, Siebke, K., and Weis, E. (1995). Imaging of chlorophyll-a-fluorescence in leaves - topography of photosynthetic oscillations in leaves of Glechoma hederacea. Photosynth. Res. 10,

15 Trissl, H. W., Gao, Y., and Wulf, K. (1993). Theoretical fluorescence induction curves derived from coupled differential equations describing the primary photochemistry of photosystem II by an exciton-radical pair equilibrium. Biophys. J. 64, van Grondelle, R., Dekker, J. P., Gillbro, T., and Sundstrom, V. (1994). Energy transfer and trapping in photosynthesis. Biochim. Biophys. Acta 1187, van Kooten, O., and Snel, J. F. H. (1990). The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25, Yakushevska, A. E., Keegstra, W., Boekema, E. J., Dekker, J. P., Andersson, J., Jansson, S., Ruban, A. V., and Horton, P. (2003). The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes. Biochemistry 42, Yamamoto, H. Y., Nakayama, T. O., and Chichester, C. O. (1962) Studies on the light and dark interconversions of leaf xanthophylls. Arch. Biochem. Biophys. 97, Yi, L., and Dalbey, R. E. (2005). Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review), Mol. Membr. Biol. 22, Zer, H., Vink, M., Shochat, S., Herrmann, R. G., Andersson, B., and Ohad, I. (2003). Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s). Biochemistry 42, Zolla, L., Rinalducci, S., Timperio, A. M., and Huber, C. G. (2002). Proteomics of lightharvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I. Plant Physiol. 130,

16 16