Examinator: Professor Lennart Eliasson, Umeå Fakultetsopponent: Amanuensis Thor Bernt Meltf, Trondheim

Transcription

1 Methods for measurements of chlorophyll fluorescence, luminiscence and photosynthesis in intact plants by Erik Sundbom AKADEMISK AVHANDLING som med tillstånd av rektorsämbetet vid Umeå universitet för erhållande av filosofie doktorsexamen, framlägges till offentlig granskning tisdagen den 9 juni 1981 kl vid Fysiologi- Botanik Hufo, seminarierum B. Examinator: Professor Lennart Eliasson, Umeå Fakultetsopponent: Amanuensis Thor Bernt Meltf, Trondheim

2 Titte: Author: Address: Abstract: Methods for measurements of chlorophyll fluorescence, luminiscence and photosynthesis in intact plants. Erik Sundbom Department of Plant Physiology, University of Umeå, S-9OI 87 Umeå, Sweden Methods were developed to study delayed light emission (luminiscence) and fluorescence changes in intact leaves of plants. Delayed light emission, detected from plants in darkness, was used to produce images of the plant leaves. The procedure was termed "phytoluminography". The use of the method is suggested for dianostic purposes at early stages of disturbances of the leaf tissues, not detectable with the naked eye. The delayed light emission is associated with the photochemistry of photosystem II and the light induced conversion and storage of energy in the thylakoid membrane system of chloroplasts. Fluorescence yield changes were induced by lowering temperature between 20 C and -20 C. The temperature induced fluorescence changes in leaves parallel the temperature induced changes in isolated chloroplasts in reaction preparations mediating photosynthetic electron transport from endogenous water splitting to added NADP. At above freezing temperatures, lowering the temperature at a constant rate of 1 C per minute caused supressed electron transport and increased fluorescence yield which were linearely dependent on the temperature change in frost resistent plants. Repeated freeze-thaw cycles between 20 C and -20 C induced variable fluorescence yield changes which were gradually depleated to F0 or Fm when the electron transport was injuried on the oxidizing or on the reduzing side of photosystem II, respectively. The temperature induced fluorescence changes were used to characterize plants with different ability to withstand freezing temperatures. The method also discriminates between plants of different frost resistance, and the method was used in screening for frost tolerance. Key words: ISBN ^ Luminiscence, delayed light emission, phytoluminography, fluorescence, in vivo chlorophyll a^ fluorescence, photosynthesis, electron transport, frost tolerance, intact plants Umeå I98I. Distributed by the Department of Plant Physiology, University of Umeå, S-9OI 87 Umeå, Sweden.

3 Methods for measurements of chlorophyll fluorescence, luminiscence and photosynthesis in intact plants by Erik Sundbom Department of Plant Physiology, University of Umeå, S-9OI 87 Umeå, Sweden Doctoral thesis ISBN ^-075-9

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5 Titte: Author: Address: Abstract: Methods for measurements of chlorophyll fluorescence, luminiscence and photosynthesis in intact plants. Erik Sundbom Department of Plant Physiology, University of Umeå, S Umeå, Sweden Methods were developed to study delayed light emission (luminiscence) and fluorescence changes in intact leaves of plants. Delayed 1ight emission, detected from plants in darkness, was used to produce images of the plant leaves. The procedure was termed "phytoluminography". The use of the method is suggested for dianostic purposes at early stages of disturbances of the leaf tissues, not detectable with the naked eye. The delayed light emission is associated with the photochemistry of photosystem II and the light induced conversion and storage of energy in the thylakoid membrane system of chloroplasts. Fluorescence yield changes were induced by lowering temperature between 20 C and -20 C. The temperature induced fluorescence changes in leaves parallel the temperature induced changes in isolated chloroplasts in reaction preparations mediating photosynthetic electron transport from endogenous water splitting to added NADP. At above freezing temperatures, lowering the temperature at a constant rate of 1 C per minute caused supressed electron transport and increased fluorescence yield which were linearely dependent on the temperature change in frost resistent plants. Repeated freeze-thaw cycles between 20 C and -20 C induced variable fluorescence yield changes which were gradually depleated to F0 or Fm when the electron transport was injuried on the oxidizing or on the reduzing side of photosystem II, respectively. The temperature induced fluorescence changes were used to characterize plants with different ability to withstand freezing temperatures. The method also discriminates between plants of different frost resistance, and the method was used in screening for frost tolerance. Key words: ISBN *» Luminiscence, delayed light emission, phytoluminography, fluorescence, in vivo chlorophyll fluorescence, photosynthesis, electron transport, frost tolerance, intact plants Umeå I98I. Distributed by the Department of Plant Physiology, University of Umeå, S Umeå, Sweden.

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7 LIST OF PAPERS This thesis is based on the following papers, which will be referred to in the text by the given Roman numerals. I Sundbom, E. and Björn, L.O Phytoluminography: Imaging plants by delayed light emission. - Physiol. Plant. ^0: 39_i*1. II Sundbom, E. and ö quist, G Temperature induced variable f1uorescence-yield changes of intact leaves. III Sundbom, E. and bquist, G Temperature induced changes of fluorescence yield and photosynthetic electron transport of isolated chloroplasts. IV Hällgren, J.-E., Sundbom, E. and Strand, M Photosynthetic responses to low temperature in Betula pubescens and Betula tortuosa. - Physiol. Plant., submitted for publication, V Sundbom, E Temperature induced fluorescence changes, a screening method for frost tolerance of potato (Solanum tuberosum L.).

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9 C O N T E N T S Page 1. INTRODUCTION 1 2. LUMINISCENCE (Delayed light emission) The slow decay component of delayed 4 1 ight emission 2.2. Phytoluminography: Imaging plants by 5 delayed light emission 3. FLUORESCENCE Fluorescence and the photochemistry 6 of photosystem Methods to induce fluorescence yield changes Temperature induced fluorescence 7 changes Freeze-thaw induced fluorescence 8 changes 4. APPLICATION OF TEMPERATURE INDUCED FLUORESCENCE CHANGES Photosynthesis and temperature induced 9 fluorescence changes in birches adapted to different temperatures 4.2. Temperature induced fluorescence changes: 9 A method in screening for frost tolerance 5. ACKNOWLEDGEMENTS REFERENCES 13

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11 - 1 - I INTRODUCTION The absorption of light by the photosynthetically active pigments of plants results in creation of singlet excitation energy. This energy conversion is the first step in photosynthesis, a complex reaction scheme of photophysical, photochemical and biochemical processes. The different processes take place at varoius time scales. The first step, light absorption, is finished within 10 ^ seconds while, carbohydrate formation, the last step may take several seconds.the processes have been divided into different eras by Kamen (1963) and are summarized by Govindjee (1975) as follows: A. Era of radiation physics, 10 ^ to 10 ^ seconds: Chi + hv > Chi Excitation (1) 9 s Chi > Chl-j. Intercrossing system (2) Chi s Chi > Chi + Chi" Excitation energy transfer (3) Chi > Chi + s g hv' Fluorescence (*») JU Chi s + T > T + Ch 1g Trapping of energy (5) y'c Chlg, chlorophyll in ground state; Chi chlorophyll in singlet excitated state; Chl^., chlorophyll triplet; T, energy trap B. Era of photochemistry, 10 to 10 seconds: D+TA" (6) DT"A ^ --- > DT+A~ > D+TA" (7) ^ D+T _A > D+TA~ (8) D, electron donator; A, electron acceptor.

12 k -2 C. Era of biochemistry, 10 to 10 seconds: D+TA" + i NADP+ + H+ > D+TA + i NADPH + i H+, reduction of NADP (9) D+TA + i H20 > DTA \ 0^ + H+, oxygen evolution (10) D+TA + ADP + P. > DTA + ATP, photophosphorylation (1 1) C NADPH + 3 ATP > (CH20) + 2 NADP+ + 3 ADP + 3 P., carbon fixation (12) At a constant absorption of light quanta by the photosynthetic pigments the excitation is influenced by the following deexcitation processes: A. Light induced electron transport over photosystem II, involving water splitting and secondary e l e c t r o n transfer redox processes. B. Radi at ion less thermal deactivation C. Radiant emission of Chi fluorescence D. Distribution of excess excitation from Chi of photosystem II to Chi of photosystem I, i.e. spillover. The Chi excitation reemitted as fluorescence varies in response to the rate of photosynthesis. It was shown by Lavorel (1959) and Clayton (19&9) that the fluorescence emission consists of a constant and a variable part. The variable part of Chi fluorescence is sensitive to the rate of electron transport over photosystem II and also to changes in the thylakoid membrane ultrastructure related to photophosphorylation.

13 - 3 - Luminiscence (delayed light emission) is caused by a back reaction of photosystem II created by the same singlet excitation that is responsible for the emission of fluorescence (promt fluorescence). The two light phenomena thus have identical spectral distribution. They are also subject to the same radiative or nonradiative decays, migration and trapping by reaction center chlorophyll (Lavorel 1968). Thus luminiscence and variable fluorescence are both associated with the functioning of photosystem II (Goedheer 1962, 1963 and Bertch 1962). The general purpose of this thesis was to study photosynthetic reactions related to the photochemistry of intact plants, using methods non- -destructive to the plant as a whole. Delayed light emission, detected in darkness, was used to produce images of the plants. The procedure was termed "phytoluminography". The use of this method is suggested for diagnostic purposes at early stages of disturbances in the leaf tissues, not detectable with the naked eye (paper I). An apparatus and a method to induce fluorescence yield changes in intact leaves through temperature changes between 20 C and -20 C are described (paper II). The temperature induced fluorescence changes at above freezing temperatures are similar in intact leaves and in isolated chloroplasts and the fluorescence yield changes are, in this temperature range, 1 inearly dependent on the rate of photosynthetic electron transport (paper III). As a means to detect differences in frost tolerance, fluorescence yield changes were also studied during repeated freeze-thaw cycles in intact leaves (paper IV and V ).

14 - k - 2. LUMINISCENCE (Delayed light emission) 2.1 The slow decay component of delayed light emission Light production by previously illuminated green plants, which lasts several minutes after the exciting light has been turned off, was discovered by Streler and Arnold (1951). The light phenomenon is known by various names: delayed light, afterglow, luminiscence and delayed fluorescence. In general, the delayed light emission is caused by a reversal of the photosynthetic conversion of light to chemical energy. Thus its intensity and decay kinetics depend on the properties of the photosynthetic apparatus. It is accepted that the delayed light emission originates from the actively functioning photosystem II, and that the slow component, lasting up to 10 minutes, correlates with the rate of the conversion and S2 > S. (Jol iot e l_ and Barbieri et^ aj_. 1970). The process can be summarized as a back reaction between the reduced primary electron acceptor Q and the oxidized primary electron donator that are produced by photosystem II during excitation: DP680A + hv' > D+P680A" D+ p680a" > DP680A + h v " hv', exciting light; h v ", delayed light emission. Of several components with different decay rates of delayed light emission (Björn 1971, Lavorel 1975), the slow component is suggested as the most sensitive indicator of deviations from normal cell function. The slow component is dependent on the properties of the thylakoids and on interactions between the chloroplasts and the rest of the cell (Björn 1971).

15 - 5 - In paper I the slow component of delayed light emission with a half- -life of the order of 1 minute was studied in intact leaves. 2.2 Phytoluminography: Imaging plants by delayed light emission One difficulty in studying the slow delayed light emission component is its extremely low intensity. As demonstrated in paper I the use of an image intensifier (Mullard XX 1063 three-stage focusing type) with a gain of about photons out per photon incident on the photocatode could detect the slow component of delayed light emission in darkness. The image produced on the image intensifier screen could then be recorded on high-speed black and white photographic film. It is also shown that thermal damage of the leaf tissues and also treatment with the photosynthetic inhibitor DCMU, 3~(3>^_dichlorophenyl)- -1,1-dimethylurea, supress the delayed light emission at the affected spots of the leaf. It was suggested that phytoluminography,imaging plants by delayed light emiss ion, might be used for diagnostic purposes to detect early stages of disturbances due to parasites, mineral deficiency, herbicides, frosts etc. in whole leaves. Later this possibility has been extensively studied, with an improved apparatus, by Björn et^ al. (1979).

16 FLUORESCENCE 3.1 Fluorescence and photochemistry of photosystem II The chlorophyll content of higher plant leaves is very high (about 10 ^ M) and at physiological temperatures chlorophyll of photosystem 11 is weakly fluorescent with a main emissionband at 685 nm and a satellite band at 7**0 nm. Probably the most studied fluorescence parameter is the relative fluorescence yield of Chi since this fluorescence yield is an index of the amount of singlet Chi excitation of photosystem 11. The amount of Chi excitation which is reemitted as fluorescence varies in response to the rate of photosynthesis. Roughly expressed, weak Chi fluorescence is typical in a situation where the photosynthetic apparatus is working undisturbed at optimal (high) rates of the photosynthetic electron transport, whereas high fluorescence is typical when the photosynthesis is weak or inhibited. However all of the Chi emission is not sensitive to the rate of photosynthetic electron transport. Lavorel (1959) and Clayton (1969) first made the distinction between fluorescence of variable (F ) and fluorescence of v constant (F ) yield. The variable part of the fluorescence emission is sensitive to the rate of electron transport over photosystem II. Thus the functioning of the electron donating and electron accepting side of photosystem II would be expected to have great influence on the variable fluorescence yield. Maximal fluorescence F ' m is obtained when the primary electron acceptor of photosystem II, Q, is fully reduced (closed traps, P680Q ) and Fq is obtained when Q. is fully oxidized (open traps, P680Q) (Butler 1978).

17 Methods to induce fluorescence yield changes Changes in fluorescence yield that reflect changes in the energy conversion efficiency of photosystem II were first reported by Kautsky and Hirsch (1931). Measurements of kinetics and yield of Fy have shown that Fy can be quenched if plants are exposed to stress e.g. chilling treatment (Smillie 1979) or prolonged water stress (Govindjee et a l. 1981). Chlorophyll fluorescence has also been used as a probe to investigate stress induced effects on the thylakoid membrane such as 1) heat induced separation of the light harvesting chlorophyll from photosystem II (Schreiber and Armond 1978), 2) temperature induced phase transitions in chilling sensitive plants (Murata et aj_. 1975) and 3) temperature jump effects (Schreiber ejt <al_. 1976, Schreiber and Berry 1977) Temperature induced fluorescence changes A fluorescence increase from Fq to F^ was induced through progressive lowering of the temperature from 20 C to -20 C, at a constant cooling rate of 1 C per minute. The method is described and evaluated in paper II and III. Lowering the temperature towards the freezing point induces a linear increase in the fluorescence yield. At the same time the electron transport between photosystem II and photosystem I is linearly inhibited (Thorne and Boardman 1971, Nolan and Smillie 1977, Smillie and Nott 1979). At the freezing point, the fluorescence shifts under influence of exothermic heat, given off when ice is formed (Melcarek and Brown 1979). Differences observed in the kinetics of the fluorescence changes were related to the structure of the leaf, i.e. mesomorphic or xeromorphic characters. Wheter applied to intact leaves or to isolated chloroplasts, the method can reveal if the photosynthetic electron

18 - 8 - transport is inhibited on the oxidizing or the reducing side of photosystem II (paper II, III). We suggest that the method provides a valuable tool for investigation of effects of environmental stress imposed on the function of photosynthesis Freeze-thaw induced fluorescence changes In successively increased freeze-thaw stress, through repeated freeze- -thaw cycles between 20 C and -20 C, principally two different schemes of reaching a totally supressed variable fluorescence were observed (paper II). The variable fluorescence was eventually supressed and invariable F or F was reached depending on the site of inhibition, o m r a F or F was reached if inhibition of the electron transport occurred o m r on the oxidizing or reducing side of photosystem II, respectively. This was shown in experiments with frost hardened and unhardened pine seedlings. Furthermore, freeze-thaw experiments with different species clearly discriminates between very frost sensitive (potato), moderately frost resistent (pea,spi nach,summer active pine) and frost resistent (hardened pine) plants.

19 APPLICATION OF TEMPERATURE INDUCED FLUORESCENCE CHANGES 4.1 Photosynthesis and temperature induced fluorescence changes in birches adapted to different temperatures Adaptation and acclimation of plant photosynthetic reactions to temperature has recently been described in detail by Berry and Björkman (1980)- In Scandinavian mountains Betula tortuosa forms the tree limit. Whether Jî. tortuosa d iffers from B. pubescens in its ability to ma inta in a high rate of photosynthesis at low temperatures was examined in paper IV. The results show that jb. tortuosa has a higher rate of net CC^ exchange at low temperatures than J3. pubescens. There was marked difference between the birches in their ability to withstand freezing, as revealed from the measurements of temperature induced fluorescence changes. It was concluded that J5. tortuosa exhibits an adaptation to the prevailing climate in the mountain region. 4.2 Temperature induced fluorescence changes: A method in screening for frost tolerance Freezing injury in plants occurs over a broad spectrum of sub-zero temperatures. The frost killing temperature depends on the actual species and on the stage of acclimation. Although there is a great diversity in the range of sub-zero temperatures that can be tolerated, there is however one common feature involved in freezing tolerance. Membrane damages are commonly inferred to be the primary effect of frost injuries (Lyons jet a_k 1979). Further, the rapidity with which injury is manifested suggests that the freezing injury is not due to metabolic disturbances, as in chi 11ing injury. In almost every country where potatoes are cultivated frosts during the growing season are a well known problem. I northern Sweden frost occurs

20 frequently during the last month before the crop Solanum tuberosum reaches maturity. Temperature induced fluorescence changes of different and closely related clones of tuberosum were measured in one freeze- thaw cycle between 20 C and -10 C (paper V ). The short term frost simulation by the freeze-thaw cycle and its influences on the fluorescence changes was compared with long term frost treatment in a climate chamber. The results of the two separately performed "blind-tests" were identical with respect to their ranking of the different clones for frost tolerance. The temperature induced fluorescence changes also monitored progressive damages to the chloroplast membranes when plants were exposed to successively lower temperatures. It was found that the differences in freezing temperature of the leaves of the different clones did not correlate with the estimated frost tolerance in the climate chamber test or fluorescence test. The fluorescence pattern observed indicates a primary effect of the freezing injury to the oxidizing side of photosystem II, since variable fluorescnece decreased to F. o An interesting discovery was made on the low temperature induced Fm fluorescence in potato. As shown in paper V, invariable Fq was induced after one freeze-thaw cycle. This situation also occurs in the first freezing period introduced, if the temperature falls below a critical value (Figure 1). This temperature is found to be -11 C for the potato cultivar Bintje, irrespective of the time (5-30 minutes) of exposure to sub-zero temperatures above -11 C. Hence the low temperatures of the first freezing period causes irreversible inhibition of the electron transport of the oxidizing side of photosystem II.

21 Fluorescence intensity (arbitrary units) 10 units Figure 1. Temperature induced fluorescence changes of an excised leaf of Solanum tuberosum L. cv. Bintje. The cool ing rate was 1 C per minute and the quantum flux density of -2-1 the exciting 1ight was 2 ye m s. The temperature of the leaf was measured by a copper-constantan thermocouple attached to the upper leaf surface. T, exciting light on; t, exciting 1ight off. The data obtained so far indicate that the transition temperature seen in the first freezing period where F decreases to F, correlate with 3 r m o the frost tolerance of four different clones of potato studied (Table 1). The frost tolerance was earlier estimated by fluorescence measurements and controlled freezing in a climate chamber (paper V ). A possibility is that low temperature weakens hydrophobic interactions in photosystem II in potato. This is known to induce structural changes of membranes which could lead to conditions of reaction center quenching of variable fluorescence (Raison et al. 1980).

22 Table 1. Transition temperature of decrease to Fq of four different clones of potato, S. tuberosum. X ± SE, n , * transition Clone no. ^. /O-x n temperature ( C) 1-11,0 ± 0, ,3 1 k -12,7 (±0,7) ,0 1 * The clone no. is identical with the clone no. used in paper V. 5. ACKNOWLEDGEMENTS I wish to express my sincere thanks to all members of the Department of Plant Physiology, University of Umeå. I am especially grateful to my supervisor and friend Docent Gunnar öquist. This work could not have been accomplished without his valuable ideas, encouragement and never failing spirit. I am also grateful to Professor Lennart Eliasson for his support and for introducing me to the field of agricultural plant physiology, and to Professor Lars Olov Björn for fruitful cooperation. The close cooperation with Doctor Jan-Erik Hällgren is deeply acknowledged. This work was supported by grants from the Swedish Natural Science Research Council, Swedish Counsil for Forestry and Agricultural Research and National Swedish Board for Technical Development.

23 REFERENCES Barbierie, G., Delosme, R. and Joliot, P. (1970). Photochem. Photobiol. 12, 187. Berry, J. and Björkman, 0. (1980). Ann. Rev. Plant Physiol. 31, 491. Bertch, W. (1962). Proc. Nat. Acad. Sei. U.S. 48, Björn, L.O. (1971). Photochemistry and Photobiology 13, 5. Björn, L.O. and Forsberg, A.S. (1979). Physiol. Plant. 47, 215. Butler, W.L. (1978). Ann. Rev. Plant Physiol. 29, 345- Clayton, R.K. (1969). Biophys. J. 9, 60. Goedheer, J.C. (1962). Biochim. Biophys. Acta 64, 29. Goedheer, J.C. (1963). Biochim. Biophys. Acta 66, 6. Govindjee, Downton, W.J.S., Fork, D.C. and Armond, P.A. (1981). Plant Sei. Lett. 20, 191. Govindjee änd Govindjee, R. (1975). In: Bioenergetics of Photosynthesis (Govindjee ed.) pp 1-50, Academic Press, New York. Joliot, P., Joliot, A., Bouges, B. and Barbieri, G. (1970). Photochem. Photobiol. 12, 287. Kamen, M.D. (1963). Primary Processes in Photosynthesis. Academic Press, New York. Kautsky, H. and Hirsch, A. (1931). Naturwissenschaften 19» 964. Lavorel, J. (1959). Plant Physiol. 34, 204. Lavorel, J. (1968). Biochim. Biophys. Acta 153, 727. Lavorel, J. (1975). In: Bioenergetics of Photosynthesis (Govindjee ed.) pp , Academic Press, New York. Lyons, J.M., Raison, J.K. and Steponkus, P.L. (1979). In: Low Temperature Stress in Crop Plants, The role of the membrane (James M. Lyons, Douglas Graham, John K. Raison Eds.) pp 1-24, Academic Press, New York. Melcarek, P.K. and Brown, G.N. (1977). Plant and Cell Physiol. 18, Melcarek, P.K. and Brown, G.N. (1979). Cryobiology 16, 69. Murata, N., Troughton, J.H. and Fork, D.C. (1975). Plant Physiol. 56, 508. Nolan, W.G. and Smillie, R.M. (1977). Plant Physiol. 59, Raison, J.K., Berry, J.A., Armond, P.A. and Pike, C.S. (1980). In: Adaption of Plants to Water and High temperature Stress (Neil C. Turner and Paul J. Kramer Eds.) pp , John Wiley and Sons, New York.

24 - I l f - Schreiber, U. and Armond, P. (1978). Biochim. Biophys. Acta 502, I38. Schreiber, U. and Berry, J.A. (1977). Planta I36, 233. Schreiber, ü., Colbow, K. and Vidaver, W. (1976). Biochim. Biophys. Acta 1 23, 249. Smillie, R.M. (1979). In: Low Temperature Stress in Crop Plants, The Role of the Membrane (James M. Lyons, Douglas Graham, John K. Raison Eds.) pp , Academic Press, New York. Smillie, R.M. and Nott, R. (1979). Plant Physiol. 63, 796. Strehler, B.L. and Arnold, W. (1951). J. Gen; Physiol. 3^, 809. Thorne, S.W. and Boardman, N.K. (1971). Biochim. Biophys. Acta 23A, 113-