Abstract

We demonstrate that picosecond time-gated fluorescence microscopy can be used to monitor subtle changes in the kinetics and spatial distribution of perturbations to the molecular and cellular structure of plant tissue caused by ultraviolet radiation. Single-molecule experiments on Photosystem II and chloroplast preparations give picosecond fluorescence decay kinetics that are similar to those obtained previously on bulk samples. For green plant leaves, localized and well-defined cellular structure is seen for normal material whereas relatively diffuse and non-specific features are seen after UV-irradiation indicating significant UV-induced rupture of the cellular structure. The changes in the chlorophyll fluorescence decay kinetics indicate uncoupling of chlorophyll molecules in the light-harvesting system leading to inhibition of energy reorganization and transfer in the antennae and subsequent exciton transfer to the reaction centers.

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  1. G. Marriott, R. M. Clegg, D. J. Arndt Jovin and T. M. Jovin, �Time-resolved imaging microscopy: fluorescence and delayed fluorescence imaging,� Biophys. J. 60, 1374-1387 (1991).
  2. J. Sytsma, J. M. Vroom, C. J. deGrauwandH. C. Gerritsen, �Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,� J. Microsc. 191, 39-51 (1998)
  3. K. Dowling, M. J. Dayel, M. J. Lever, P. M. W. French, J. D. Hares and A. K. L. Dymoke-Bradshaw, �Fluorescence lifetime imaging with picosecond resolution for biomedical applications,� Opt. Lett. 23, 810-812 (1998).
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  11. J. M. Anderson, Y. -I. Park and W. S. Chow, �Photoinactivation and photoprotection of Photosystem II in nature,� Physiol. Plantarum 100, 214-223 (1997).
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  14. S. A. Post, P. B. Lukins, P. J. Walker andA.W. D. Larkum, �The effects of ultraviolet radiation on P680+reduction in PS II core complexes measured in individual S-states and during repetitive cycling of the oxygen-evolving complex,� Photosynth. Res. 49, 21-27 (1996).
  15. D. A. Berthold, G. T. Babcock and C. A. Yocum, �A highly resolved oxygen-evolving Photosystem II preparation from spinach thylakoid membranes,� FEBS Lett. 134, 231-234 (1981).
  16. D. Walker, �Mechanical isolation of chloroplasts� in �The use of oxygen electrodes and fluorescent probes in simple measurements of photosynthesis�, (Oxygraphics, Sheffield, 1987) 107-114.
  17. V. Barzda, C. J. de Grauw, J. M. Vroom, F. J. Kleima, R. van Grondelle, H. van Amerongen and H. C. Gerritsen, �Fluorescence lifetime heterogeneity in aggregates of LHC II revealed by time-resolved microscopy,� Biophys. J. 81, 538-546 (2001).
  18. D. J. Blubaugh, M. Atamian, G. T. Babcock, J. H. Goldbeck and G. M. Cheniae, �Photoinhibition of hydroxylamine-extracted Photosystem II membranes: identification of the sites of photodamage,� Biochemistry 30, 7586-7597 (1991).

Ambio (1)

M. M. Caldwell, A. H. Teramura, M. Tevini, J. F. Bornman, L. O. Bjorn and G. Kulandaivelu, �Effects of increased solar ultraviolet radiation on terrestrial plants,� Ambio 24, 166-173 (1995).

Appl. Phys. Lett. (1)

M. Straub and S. W. Hell, �Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope,� Appl. Phys. Lett. 73, 1769-1771 (1998).

Biochemistry (2)

I. Vass, L. Sass, C. Spetea, A. Bakou, D. F. Ghanotakis and V. Petrouleas, �UV-B induced inhibition of Photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor- and acceptor-side components,� Biochemistry 35, 8964-8973 (1996).

D. J. Blubaugh, M. Atamian, G. T. Babcock, J. H. Goldbeck and G. M. Cheniae, �Photoinhibition of hydroxylamine-extracted Photosystem II membranes: identification of the sites of photodamage,� Biochemistry 30, 7586-7597 (1991).

Biochim. Biophys. Acta (1)

T. A. Roelofs, M. Gilbert, V. A. Shuvalov and A. R. Holzwarth, �Picosecond fluorescence kinetics of the D1/D2- cytb559 Photosystem II reaction center complex energy transfer and primary charge separation processes,� Biochim. Biophys. Acta 1060, 237-244 (1991).

Biophys. J. (4)

T. A. Roelofs, C. -H. Lee and A. R. Holzwarth, �Global target analysis of picosecond chlorophyll fluorescence kinetics from pea chloroplasts. A new approach to the characterization of the primary processes in Photosystem II alpha- and beta-units,� Biophys. J. 61, 1147-1163 (1992).

V. Barzda, V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Ameroagen, R. van Grondelle and L. Valkunas, �Singlet-singlet annihilation kinetics in aggregates and trimers of LHC II,� Biophys. J. 80, 2409-2421 (2001).

G. Marriott, R. M. Clegg, D. J. Arndt Jovin and T. M. Jovin, �Time-resolved imaging microscopy: fluorescence and delayed fluorescence imaging,� Biophys. J. 60, 1374-1387 (1991).

V. Barzda, C. J. de Grauw, J. M. Vroom, F. J. Kleima, R. van Grondelle, H. van Amerongen and H. C. Gerritsen, �Fluorescence lifetime heterogeneity in aggregates of LHC II revealed by time-resolved microscopy,� Biophys. J. 81, 538-546 (2001).

FEBS Lett. (1)

D. A. Berthold, G. T. Babcock and C. A. Yocum, �A highly resolved oxygen-evolving Photosystem II preparation from spinach thylakoid membranes,� FEBS Lett. 134, 231-234 (1981).

J. Microsc. (1)

J. Sytsma, J. M. Vroom, C. J. deGrauwandH. C. Gerritsen, �Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,� J. Microsc. 191, 39-51 (1998)

Opt. Lett. (1)

Photobiochem. & Photobiophys. (1)

J. F. Bornman, L. O. Bjorn and H. E. Akerlund, �Action spectrum for inhibition by UV radiation of Photosystem II activity in spinach-oleracea thylakoids,� Photobiochem. & Photobiophys. 8, 305-314 (1984).

Photochem. Photobiol. (1)

G. Renger,M. Volker, H. -J. Eckert, R. Fromme, S. Hohm-Veit and P. Graeber, �On themechanismof Photosystem II deterioration by UV-B irradiation,� Photochem. Photobiol. 49, 97-106 (1989).

Photosynth. Res. (1)

S. A. Post, P. B. Lukins, P. J. Walker andA.W. D. Larkum, �The effects of ultraviolet radiation on P680+reduction in PS II core complexes measured in individual S-states and during repetitive cycling of the oxygen-evolving complex,� Photosynth. Res. 49, 21-27 (1996).

Photosynthetica (1)

C. Buschmann, G. Langsdorf and H. K. Lichtenhaler, �Imaging of the blue, green and red fluorescence emission of plants: An overview,� Photosynthetica 38, 483-491 (2000).

Physiol. Plantarum (1)

J. M. Anderson, Y. -I. Park and W. S. Chow, �Photoinactivation and photoprotection of Photosystem II in nature,� Physiol. Plantarum 100, 214-223 (1997).

Other (1)

D. Walker, �Mechanical isolation of chloroplasts� in �The use of oxygen electrodes and fluorescent probes in simple measurements of photosynthesis�, (Oxygraphics, Sheffield, 1987) 107-114.

Supplementary Material (1)

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