Abstract
A newly-developed field-portable multi-flash kinetic fluorimeter for measuring the kinetics of the microsecond to millisecond reactions of the oxidizing and reducing sides of photosystem 2 in leaves of intact plants is described and demonstrated. The instrumental technique is a refinement of that employed in the ‘double-flash’ kinetic fluorimeter (Joliot 1974 Biochim Biophys Acta 357: 439–448) where a low-intensity short-duration light pulse is used to measure the fluorescence yield changes following saturating single-turnover light pulses. The present instrument uses a rapid series of short-duration (2 μs) pulses to resolve a complete microsecond to millisecond time-scale kinetic trace of fluorescence yield changes after each actinic flash. Differential optics, using a matrix of optical fibers, allow very high sensitivity (noise levels about 0.05% Fmax) thus eliminating the need for signal averaging, and greatly reducing the intensity of light required to make a measurement. Consequently, the measuring pulses have much less actinic effect and an entire multi-point trace (seven points) excites less than 1% of the reaction centers in a leaf. In addition, bu combining the actinic and measuring pulse light in the optical fiber network, the tail of the actinic flash can be compensated for, allowing measurements of events as rapidly as 20 μs after the actinic flash. This resolution makes practical the routine measurement of the microsecond turnover kinetics of the oxygen evolving complex in leaves of intact plants in the field. The instrument is demonstrated by observing flash number dependency and inhibitor sensitivity of the induction and decay kinetics of flash-induced fluorescence transients in leaves of intact plants. From these traces the period-two oscillations associated with the turnover of the two-electron gate and the period-four oscillations associated with the turnover of the oxygen evolving complex can be observed. Applications of the instrument to extending our knowledge of chloroplast function to the whole plant, the effects on plants of environmental stress, herbicides, etc, and possible applications to screening of mutants are discussed.
Similar content being viewed by others
Abbreviations
- DCMU:
-
3-(3,4-Dichlorophenol)-1,1-dimethylurea
- PS 2:
-
photosystem 2
- PS 1:
-
photosystem 1
- P680 :
-
primary electron donor of the PS 2 reaction center
- QA :
-
primary acceptor quinone of PS 2
- QB :
-
secondary acceptor quinone of PS 2
- CCCP:
-
carbonyl cyanide-m-chlorophenylhydrazone
- Yz :
-
donor to P680 +
- F0 :
-
level of fluorescence with all PS 2 centers open
- Fmax :
-
maximum level of fluorescence with all PS 2 centers closed
- P680QA :
-
Open reaction centers with P680 reduced and QA oxidized (low fluorescence)
- P680QA - :
-
Closed reaction centers, in which P680 is reduced (high fluorescence)
- P680 +QA - :
-
Closed reaction centers, in which P680 is oxidized (low fluorescence)
References
Babcock GT, Blankenship RE and Sauer K (1976) Reaction kinetics for positive charge accumulation on the water side of chloroplast photosystem II. FEBS Lett 61: 286–289
Bowes JM and Crofts AR (1980) Binary oscillations in the rate of reoxidation of the primary acceptor of photosystem II. Biochim Biophys Acta 590: 373–384
Brettel K, Schlodder E and Witt HT (1984) Nanosecond reduction kinetics of photooxidized chlorophyll-a II (P-680) in single flashes as a probe for the electron pathway, H+ release and charge accumulation in the O2-evolving complex. Biochim et Biophys Acta 766: 403–415
Butler WL, Visser JWM and Simons HL (1979) The back reaction in the primary electron transfer couple of photosystem II of photosynthesis. Biochim Biophys Acta 325: 539–545
Crofts AR and Wraight CA (1983) The electrochemical domain of photosynthesis. Biochim et Biophys Acta 726: 149–185
Delosme R (1971) New results about chlorophyll fluorescence in vivo. In: Forti G, Avron M and Melandri A (eds) Proceedings of the 2nd International Congress on Photosynthesis. Junk, The Hague
Deprez J, Dobek A, Geacintov NE, Paillotin G and Breton J (1983) Probing fluorescence induction in chloroplasts on a nanosecond time scale utilizing picosecond laser pulse pairs. Biochim Biophys Acta 725: 444–454
Diner BA and Joliot P (1976) Effect of transmembrane electric field on the photochemical and quenching properties of photosystem II in vivo. Biochim Biophys Acta 423: 479–498
Duysens LNM and Sweers HE (1953) In: Studies on Microalgae and Photosynthetic Bacteria, pp 353–372. University of Tokyo Press, Toyko
Forbush B, Kok B and McGloin B (1971) Cooperation of charges in photosynthetic O2 evolution—II. Damping of flash yield oscillation, deactivation. Photochem Photobiol 14: 307–321
Ghanotakis DF, Yerkes CT and Babcock GT (1982) The role of reagents accelerating the deactivation reactions of water splitting enzyme system Y (ADRY Reagents) in destabilizing high potential oxidizing equivalents generated in chloroplast photosystem II. Biochim Biophys Acta 682: 21–31
Hoagland DR and Arnon D (1950) The water-culture method for growing plants without soil. California Agricultural Experimental Station Circular 347. College of Agriculture, University of California, Berkeley, California
Joliot A (1974) Effect of low temperature (-30 to -60°C) on the reoxidation of the photosystem II primary electron transfer in the presence and absence of 3(3,4-dichlorophenyl)-1,1-Dimethyl-Urea. Biochim Biophys Acta 357: 439–448
Joliot P, Bennoun P and Joliot A (1973) New evidence supporting energy transfer between photosynthetic units. Biochim Biophys Acta 305: 317–328
Joliot P, Joliot A, Bouges B and Barbieri G (1971) Studies of system II photocenters by comparative measurements of luminescence, fluorescence, and oxygen emission. Photochem Photobiol 14: 287–305
Joliot A and Joliot P (1964) Etude cinetique de la reaction photochimique liberant l'oxygen au cours de la photosynthese. CR Acad Sci Paris 143: 4622–4625
Kok B, Forbush B and McGloin M (1970) Cooperation of charges in photosynthetic O2 evolution-1. A linear four step mechanism. Photochem Photobiol 11: 457–475
Kramer DM and Crofts AR (1989) Activation of the chloroplast ATPase measured by the electrochromic shift in leaves of intact plants. Biochim Biophys Acta 976: 28–41
Mauzerall D (1972) Light-induced fluorescence changes in Chlorella and the primary photoreactions for the production of oxygen. Proc Natl Acad Sci USA 69: 1358–1362
Renger G (1969) Reactions of CCCP in photosynthesis on an intermediate between chlorophyll-a II and water. Naturwis-senshaften 56: 370–372
Robinson HH (1986) Non-invasive measurements of photosystem II reactions in the field using flash fluorescence. In: Gensler WG (ed) Advanced Agricultural Instrumentation, pp 92–106. Springer-Verlag, New York
Robinson HH and Crofts AR (1983) Kinetics of proton uptake and the oxidation-reduction reactions of the quinone acceptor complex of PS II from pea chloroplasts. In: Sybesma C (ed) Advances in Photosynthesis Research, pp 477–480. Nijhoff/Junk, Publ, The Hague
Robinson H and Crofts AR (1987) Kinetics of the changes in oxidation-reduction state of the acceptors and donors of photosystem II in pea thylakoids measured by flash fluorescence. In: Biggins J (ed) Progress in Photosynthesis, pp 429–432
Robinson HH, Eaton-Rye JJ, van Rensen JJS and Govindjee (1984) The effects of bicarbonate depletion and formate incubation on the kinetics of oxidation-reduction reactions of the photosystem II quinone acceptor complex. Z Naturforsch 39: 382–385
Robinson H, Golden S, Brusslan J and Hasselkorn R (1987) Functioning of photosystem II in mutant strains of the cyanobacterium Anacystis Nidulans R2. In: Biggins J (ed) Progress in Photosynthesis, pp 825–828. Martinus Nijhoff, Publ
Sonneveld A, Rodemaker H and Duysens LNM (1979) Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680+ of photosystem II. Biochim Biophys Acta 548: 536–551
Sundblad L-G (1988) Dark reduction of QA in intact barley leaves as an effect of lowered CO2 concentration monitored by chlorophyll a luminescence and chlorophyll a F0 dark fluorescence. Biochim Biophys Acta 936: 429–434
Taoka S, Robinson HH and Crofts AR (1983) Kinetics of the reactions of the two-electron gate of photosystem II: Studies on the competition between plastoquinone and inhibitors. In: Inoue Y, Crofts AR, Govindjee, Murata N, Renger G and Satoh K (eds) The Oxygen Evolving System of Photosynthesis, pp 369–381. Academic Press, Japan
Wollman F-A (1978) Determination and modification of the redox state of the secondary acceptor of photosystem II in the dark. Biochim Biophys Acta 503: 263–273
Yerkes CT (1981) A kinetic model of the oxidizing side of chloroplasts photosystem II using time resolved EPR spectroscopy. PhD Thesis. Michigan State University, USA
Yerkes CT, Babcock GT and Crofts AR (1983) A trisinduced change in the midpoint potential of Z, the donor of photosystem II, as determined by the kinetics of the back reaction. FEBS Lett 158: 359–363
Yerkes CT, Kramer DM, Fenton JM and Crofts AR (1990) UV-photoinhibition: Studies in vitro and in intact plants. In: Press in the Proceedings of the 8th International Congress on Photosynthesis, Stockholm
Zankel KL (1973) Rapid fluorescence changes observed in chloroplasts. Their relationship to the O2 evolving system. Biochim Biophys Acta 325: 138–148
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kramer, D.M., Robinson, H.R. & Crofts, A.R. A portable multi-flash kinetic fluorimeter for measurement of donor and acceptor reactions of Photosystem 2 in leaves of intact plants under field conditions. Photosynth Res 26, 181–193 (1990). https://doi.org/10.1007/BF00033131
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00033131