PLANT PHOTOSYNTHESIS AND ITS RELATIONS TO ENVIRONMENTAL CONDITIONS

Scientific Project for 2003-2007 funded by Estonian Government

Summary

Factors and processes controlling the photosynthetic rate of wild type and transgenic plants are investigated under normal and stress conditions in the laboratory and in natural plant communities using contemporary biophysical and physiological methods. In focus are such parameters of the photosynthetic machinery as photosystems II and I, interphotosystem electron transport through Cytochrome b6f and kinetics/activity of Rubisco.

1. Introduction

Photosynthesis is the only process in the life cycle where organic matter is created from the inorganic and, as such, its importance does not need comments, especially considering the unexpected slow progress in thermonuclear energetics. Plant photosynthesis determines our life quality through food, energy and environment, therefore, scientific investigation of photosynthesis and related processes is an important area of science. Photosynthesis research is interdisciplinary, beginning from the biophysics of picosecond processes , through molecular biology and physiology to ecology and climatology. Science in the chair of Plant Physiology of the Institute of Molecular and Cell Biology widely covers this range. The project described here extends from biophysics to physiology, with applications in instrument design and technology.

2. Scientific description of the project

2.1. Non-photochemical quenching of excitation in Photosystem II

Photosynthesis begins with absorption of a photon on an antenna chlorophyll, followed by migration of excitation to a center Chl, which becomes oxidized sending the e- to the acceptor. Losses of excitation can occur on the pathway, first due to natural thermal conversion, second due to biologically controlled competing "non-photochemical" quenchers. The biological regulation of the latter seems to be imperfect, because under low light they should be completely switched off, in many cases this does not happen, especially under stress. As a result, additional losses of quanta decrease the practical quantum efficiency of photosynthesis. We have investigated the mechanism of non-photochemical quenching with an aim to know, what are the minimum possible losses and when and why the additional quenchers stay on (Laisk and Oja 1998, Oja and Laisk 1998, Laisk and Oja 2000, Oja and Laisk 2000). The following question has emerged from these works: is the slowly reversible quenching (photoinhibition) caused by the prolonged stay-on of the reversible "energy dependent" quenching under stress (the same mechanism but interfered control) or one has to do with another mechanism? If both hypotheses are correct, then what is their relative importance?

The following investigations will be undertaken under this problem.

  1. How spectrally selective is the non-photochemical quencher in the antenna? To answer the question we shall measure the quantum yield of PSII (O2 evolution) in white light and under different monochromatic light beginning from the FarRed (720 nm). The spectral selectivity of the quencher will show which Chl molecules are in contact with the quencher.
  2. We shall also investigate the onset of photoinhibition in the presence and absence of the fast-relaxing "energy dependent" quenching. For this we shall use transgenic plants with limited capacity for non-photochemical quenching, but also we shall apply strong single-turnover flashes to create photoinhibition. These flashes supply a big quantum dose but are too short to create "energy dependent" quenching. These experiments are expected to show how much the energy dependent quenching can protect against photoinhibition.
  3. We shall investigate plants under conditions when the productivity of leaf photosynthesis exceeds the needs of the plant for assimilates. We shall see whether the down-regulation of photosynthesis under these conditions is still related to the same "energy dependent" quenching or it is related to the destructive type of photoinhibition.

2.2. Antenna cross-sections of photosystems II and I

Relative optical cross-sections of PSII and PSI antennae can be found from parallel measurements of CO2 fixation, Chl fluorescence and 820 nm absorptance (Eichelmann and Laisk 2000, Laisk et al. 1999). This method is applied for the investigation of the regulation of antenna cross-section.

1. The adjustment of PSII and PSI antennae to changes in spectral composition of light (state-transition). We induce the state transition by illuminating leaves with red or far-red light. We show quantitatively how much Chl is detached from PSII. The physiological importance of the state transition will be analyzed.

2. Using the same method, the antenna cross-sections will be measured in C4 plants. In these plants the PSI antenna must theoretically be double the PSII antenna in order to support the ATP production needed for CO2 transport. The actual ratio of antennae in C4 plants will allow to draw conclusions about the energetics and mechanism of CO2 transport in these plants.

2.3. Control of electron transport from PSII to PSI and through PSI.

This section of the work is partially funded by Estonian Science Foundation, Grant 5236)

Whilst PSII can be investigated using Chl fluorescence, this is not possible for PSI, because PSI antenna emits only weak and constant fluorescence. The state of PSI may investigated using leaf transmission at 820 nm, which reflects the oxidation state of P700, the donor pigment of PSI. Unfortunately, the spectra of oxidized plastocyanin (PC+) and Fd overlap with the spectrum of P700+, what complicates the interpretation of the 820 nm signal. We analyze this signal by gradually oxidizing the PSI donor side under Far-Red Light (FRL). We have worked out a mathematical algorithm for deciphering the 820 nm signal, which allows us to rapidly get information about the oxidation state of the donor side carriers of PSI, such as P700, PC and Cyt f in an intact photosynthesizing leaf (Oja et al, 2003).

The rate-limiting step of electron transport is the Cytochrome b6f (Cyt b6f) between the two photosystems. Its turnover rate is subject to regulation, but the mechanism and principles of this regulation are not yet clear. Of special importance is the presence and regulation of the cyclic electron transport: when and via what pathway electrons return to the donor side of PSI, is it Cyt b6f? How big is the role of direct back-reaction from reduced acceptor side carriers? In these experiments we use specially designed computer-operated shutters that generate short pulses of light or darkness (Eichelmann et al. 2000). The logic of these experiments is briefly the following.

  1. PSI redox-titration under FRL allows us to find the pool sizes of the donor side carriers and to derive the algorithm for the deconvolution of the 820 nm signal. The algorithm allows us to find the amount and the redox state of the donor side e- carriers.
  2. Using the algorithm we are able to measure the actual speed of PSI electron transport during photosynthesis. We measure the 820 nm signal characterizing the speed of post-illumination re-reduction of the PSI donor side carriers previously oxidized in the light. From the speed of the post-illumination transient of the 820 nm signal we calculate the speed of electron arrival at the PSI donor side. If this is faster than the speed of electron transport through PSII (which is measured using Chl fluorescence) the difference presents the speed of the cyclic electron flow.
  3. It is known that electrons enter the interphotosystem space spontaneously from the PSI acceptor side pools related to the carbon reduction cycle (CRC). This process is easily visible when FRL is interrupted and the previously oxidized interphotosystem carriers become reduced in the dark. We investigate, how the spontaneous reduction of interphotosystem carriers is related to the accumulation of assimilates in the leaf and in the CRC. We propose that the rate of PSI electron transport is regulated by acceptor side reduction and this process is used for the control of the photosynthetic rate of the plant when photosynthesis tends to exceed the rate of assimilate utilization by the plant.
  1. In reference experiments we also compare the P700+ re-reduction speed in wild type and Cyt b6f deficient transgenic tobacco plants, with the aim to investigate how the amount of Cyt b6f is related to its turnover rate, i.e., will the remaining Cyt b6f turn over faster when the amount decreases?

2.4. Relationships between the amount and the activity of Rubisco in intact leaves.

The enzyme of CO2 fixation in C3 plants is RuBP carboxylase-oxygenase (Rubisco). We have investigated the relationships between the amount of Rubisco and the rate of photosynthesis in laboratory-grown intact leaves (Eichelmann and Laisk 1998, Eichelmann and Laisk 1999). From these experiments we concluded that the actual activity of Rubisco is not equivalently related to its amount. Evidently, the synthesis of Rubisco protein is determined by one group of factors, while its activation is determine by other factors. In this part of the project we investigate the relationships between the actual in vivo Rubisco activity and its amount in differently grown plants in the laboratory and also in plants grown outdoors. We expect to elucidate the relationships between the factors controlling the synthesis of the protein (proteomics) and the factors controlling the activity of the enzyme (metabolics).

  1. Relationships between the amount and activity of Rubisco in plants grown on different nitrogen supply. By growing plants on solutions with different N content we change the N content in leaves. We check the hypothesis according to which the apparent specific activity of Rubisco will increase with decreasing the amount of the enzyme. In case of the affirmative answer we shall get the in vivo maximum kcat of the fully activated enzyme. In case of negative answer we shall look for the parallel factors that may determine the actual activity of the enzyme (most likely, factors activating the Rubisco activase).
  2. The same approach on leaves grown in natural plant communities and on leaves of different age during their development.

    2.5. The effect of tropospheric ozone on plants

    The investigations of ozone as an abiotic stress factor have introduced us to a wider problem: what is the mechanism of the oxydative burst induced in living cells by a pathogen or a stress factor? What is the ability of the cell to generate the active oxygen species (O2· - , H2O2, · OH) during the oxydative burst?

    1. We formulate the existing knowledge and data about the peroxidase-based oxydative burst as a mathematical model, with an aim to quantitatively describe and analyse the experimental results obtained with cell suspensions and intact leaves.

2. We continue the experiments where plants are exposed under different concentration and dose of O3 . We measure CO2 and O2 exchanege, Chl fluorescence and 820 nm transmission signal, applying all experimental possibilirties of our photosynthesis laboratory. Applying this set of advanced techniques we expect to elucidate, which parts of the photosynthetic machinery are the most sensitive to elevetaed O3 .

    2.6. The use of transgenic plants in photosynthesis research

    We do not make transgenic plants in our laboratory, but use the material created in other laboratories. Our sophisticated experimental system allows us to investigate the responses of these plants in much greater details than is possible in other laboratories. For example, we have used the Cyt b6f deficient tobacco mutant created in Australian National University ( Eichelmann and Laisk 2000, Eichelmann et al. 2000). Work is in progress with the Rubisco deficient mutant from the same laboratory of ANU.

    1. Using the Rubisco deficient tobacco mutant (a gift from the Australian National University) we measure how the specific activity of the enzyme (its apparent kcat in vivo) changes with decreasing amount of the enzyme. From this experiment we expect to determine the true maximum kcat in vivo of the fully active Rubisco protein. As explained above (2.4.) we expect to find an answer to the question, what determines the actual in vivo activity of such a metabolically important enzyme as Rubisco, i.e., how big is the relative role of metabolics compared with genomics and proteomics?

    2.7. Mathematical modeling of photosynthesis

    Though the mathematical analysis of experimental results is applied continuously in our work, one line of our research is directed toward the completion of the mathematical model of the whole photosynthetic process. A characteristic feature of these models is the heuristic outcome, explaining previously not understood phenomena. We have published a mathematical model of C4 photosynthesis that explained the actual mechanism of CO2 concentration in the bundle sheath of NADP-Malic enzyme type C4 plants (Laisk and Edwards, 2001). We are about to complete a model of C3 photosynthesis that finally will explain the nature of photosynthetic oscillations.

    2.8. Instrument design and commercialisation

    The whole project has become possible thanks to unique self-made instruments and computer programming, allowing us to measure what colleagues in other laboratories cannot measure (Laisk and Oja, 1998). The measurement system created and manufactured in our laboratory is unique by its flexibility, preciseness, speed of measurement and user-friendliness (computerization). For example, even the most complicated experimental routines involving changing light intensity, CO2 and O2 concentration, multiple-turnover pulsing and single-turnover flashing are fully preprogrammed and computer-operated. Further development of this side of the project is heading toward commercialization of the full system and design of smaller instruments for wider market.

    3. Laboratory equipment basis and personnel

    In our chair of plant physiology of the Institute for Molecular and Cell Biology, as a result of long lasting efforts (since 1965), a top class has been achieved in the research of photosynthesis of intact leaves using biophysical and kinetic methods. The strongest side of our work is the interpretation of experimental results using mathematical models and original instrument design. The emphasis has been put on kinetic investigation of factors and processes determining (and limiting) the rate of photosynthesis under normal and stress conditions, beginning from energy conversion in photosystems and electron transport to carbon dioxide assimilation and end product synthesis. Thanks to long experience the principal scientist has wide expertise from biophysics to physiology (Prof. A. Laisk is one of the most frequently cited scientists in Tartu University). Senior scientist Vello Oja is an absolute virtuoso on kinetic experimentation and mathematical data processing. Scientist Hillar Eichelmann has led several laborious projects (e.g. the COPERNICUS project BETULA on the influence of elevated CO2 and O3 on birch photosynthesis). Scientist Irina Bichele is an expert on the influence of ozone on photosynthesis. Associate professor Dr. Evi Padu has worked out an SDFS PAGE method for determining the absolute amount of Rubisco in leaves. Recently a German scientist Dr. Katja Hüve joined our laboratory and she works on responses of photosynthesis to high-temperature and ozone stress.

    Thanks to instrument design and relatively good financing by Estonian Government and European Council (the COPERNICUS grant during 1999-2001), our laboratory is one of the best-equipped for the research of leaf photosynthesis. The leading figure in instrument design is Dr. Bakhtier Rasulov, a Tajik scientist who is now working for Tartu University. The most artist hand-work is done by engineer Heikko Rämma.

    4. Prospects and applications

    The prospect of photosynthesis research is still aimed at understanding, what determines and limits the rate of photosynthesis? The limiting role of one or another rate-limiting process may become crucial in different stress situations. Understanding, what are the limiting processes depending on light gradient in a canopy, at extremely high or low temperatures, elevated concentrations of atmospheric pollutants, excess or deficient water supply etc. it is possible to choose the most optimal species for cultivation or avoid the most lethal combinations of stresses. On the other hand, contemporary biotechnology is able to change the plant genome in a desired manner, the role of the fundamental research is to preplan the genomic interference and to test the practical result of it. The commercialization of the advanced scientific instrumentation is one of the milestones on the way to science-based economy in Estonian Republic.

5. Current Contents-cited papers published under the project 1998-2003 (august)
  1. Laisk, A and Oja, V. (1998) Dynamic gas exchange of leaf photosynthesis. Measurement and interpretation. CSIRO Publishing, Australia (monograafia).
  2. Eichelmann, H. and Laisk, A. (1998) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content in leaves, assimilatory charge and mesophyll conductance. In Photosynthesis: Mechanisms and Effects, Volume V. Edited by Garab, G. pp. 3383-3386, Kluwer Acad. Publ., Dordrecht/Boston/London.
  3. Kollist, H., Moldau, H., Rasmussen, S.K. and Mortensen, L. (1998) The effect of reduced light and ozone on apoplastic ascorbate and photosynthesis in the leaves of barley. In Photosynthesis: Mechanisms and Effects, Vol. IV. Edited by Garab, G. pp. 2733-2736, Kluwer Acad. Publ., Dordrecht/Boston/London.
  4. Laisk, A. and Oja, V. (1998) Oxygen evolution, chlorophyll fluorescence and electron transport through photosystem II in light pulses: acceptor resistance is dependent on nonphotochemical excitation quenching. In Photosynthesis: Mechanisms and Effects. Edited by Garab, G. pp. 1219-1222, Kluwer Acad. Publ., Dordrecht/Boston/London.
  5. Oja, V. and Laisk, A. (1998) Oxygen evolution, chlorophyll fluoprescence and electron transport through photosystem II in light pulses: quantification of the donor resistance in leaves. In Photosynthesis: Mechanisms and Effects. Edited by Garab, G. pp. 1215-1218, Kluwer Acad. Publ., Dordrecht/Boston/London.
  6. Ruuska, S., Andrews, J.T., Badger, M.R., Hudson G.S., Laisk, A., Price, D. and von Caemmerer, S. (1998) The interplay between limiting processes in C3 photoynthesis studied by rapid-response gas exchange using transgenic tobacco impaired in photosynthesis. Austral. J. Plant Physiol. 25: 859-870
  7. Laisk, A. and Edwards, G.E. (1998) Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in bundle sheath. Planta 205: 632-645
  8. Laisk, A., Rasulov, B.H. and Loreto, F. (1998) Thermoinhibition of photosynthesis as analyzed by gas exchange and chlorophyll fluorescence. Russian J. Plant Physiol. 45: 412-421
  9. Moldau, H. (1998) Hierarchy of ozone scavenging reactions in the plant cell wall. Physiol. Plantarum 104: 617-622
  10. Moldau, H., Bichele, I. and Hüve, K. (1998) Dark-induced ascorbate deficiency in leaf cell walls increases plasmalemma injury under ozone. Planta 207: 60-66
  11. Ranieri, A., Castagna, A., Padu, E., Moldau, H., Rahi, M. and Soldatini, G.E. (1998) The decay of O3 through direct reaction with cell wall ascorbate is not sufficient to explain the different degrees of O3-sensitivity in two poplar clones. J. Plant Physiol. 154: 250-255
  12. Eichelmann, H. and Laisk, A. (1999) Ribulose-1,5-bisphosphate carboxylase/oxygenase content, assimilatory charge and mesophyll conductance in leaves. Plant Physiol. 119: 179-189
  13. Moldau, H. (1999) Ozone detoxification in the mesophyll cell wall during a simulated oxidative burst . Free Radical Res. 31: S19-34
  14. Niinemets, Ü., Oja, V., Kull, O. (1999) Shape of leaf photosynthetic electron transport versus temperature response curve is not constant along canopy light gradients in temperate deciduous trees. Plant Cell and Environm., 22, 1497-1513
  15. Oja, V., Savtchenko, G., Burkhard, J. and Heber, U. (1999) pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves. Planta 209: 239-249
  16. Padu, E. (1999) Apoplastic peroxidases, ascorbate and lignification in relation to nitrate supply in wheat stem. J. Plant Physiol, 154: 576-583
  17. Bichele I., Moldau H. and Padu E. (2000) Estimation of plasmalemma conductivity to ascorbic acid in intact leaves under ozone. Physiol. Plantarum 108: 405-412
  18. Kollist H., Moldau H., Mortensen L., Rasmussen S.K., Jörgensen, L.B. (2000) Ozone flux to plasmalemma in barley and wheat is controlled by staomata rather than by direct reaction of ozone with cell ascorbate. J. Plant Physiol. 156: 645-651.
  19. Eichelmann H, Laisk A (2000) Cooperation of photosystems II and I in leaves as analyzed by simultaneous measurements of chlorophyll fluorescence and transmittance at 800 nm. Plant Cell Physiol. 41(2): 138-147.
  20. Eichelmann H, Price D, Badger M, Laisk A (2000) Photosynthetic parameters of leaves of wild-type and Cyt b6/f deficient transgenic tobacco studied by CO2 uptake and transmittance at 800 nm. Plant Cell Physiol 41(4): 432-439
  21. Laisk, A., Oja V. (2000) Alteration of Photosystem II properties with non-photochemical excitation quenching. Phil. Trans. R. Soc. Lond. (2000) 355: 1405-1418
  22. Laisk A, Oja V (2000) Electron transport through photosystem II in leaves during light pulses: acceptor resistance increases with nonphotochemical excitation quenching. Biochim. Biophys. Acta 1460, 255-267
  23. Oja, V. Laisk, A. (2000) Oxygen yield from single turnover flashes in leaves: non-photochemical excitation quenching and the number of active PSII. Biochim. Biophys. Acta, 1460, 291-301.
  24. Laisk A, Edwards GE (2000) A mathematical model of C4 photosynthesis: the mechanism of concentrating CO2 in NADP-malic enzyme type species. Photosynthsis Research, 66, 199-224
  25. Niinemets, Ü., Kull, O (2001) Sensitivity of photosynthetic electron transport to photoinhibition in a temperate deciduous forest canopy: photosystem II openness, non-radiative energy dissipation and excess irradiance under field conditions. Tree Physiology 21, 899-914
  26. Niinemets, Ü. (2001) Climatic controls of leaf dry mass per area, density, and thickness in trees and shrubs at the global scale. Ecology 82:453-469.
  27. Kollist, H. Moldau, H., Oksanen E., Vapaavuori, E. (2001) Ascorbate transport from the apoplast to the symplast in intact leaves. Physiologia Plantarum, 113, 377-384
  28. Niinemets, Ü., D.S. Ellsworth, A. Lukjanova and M. Tobias. 2001. Site fertility and the morphological and photosynthetic acclimation of Pinus sylvestris needles to light. Tree Physiology 21: 1231-1244.
  29. Peterson, R., Oja, V. Laisk, A. (2001) Chlorophyll fluorescence at 680 and 730 nm and its relationship to photosynthesis. Photosynthesis Research, 70, 185-196
  30. Laisk, A. Oja, V. Rasulov, B. Rämma, H. Eichelmann, H. Kasparova, I. Pettai, H. Padu, E. Vapaavuori, E. (2002) A computer-operated routine of gas exchange and optical measurements to diagnose photosynthetic apparatus in leaves. Plant Cell Env. 25: 923-943
  31. Niinemets, Ü. and S. Fleck. (2002). Petiole mechanics, leaf inclination, morphology, and investment in support in relation to light availability in the canopy of Liriodendron tulipifera. Oecologia 132:21-33.
  32. Niinemets, Ü., K. Hauff, N. Bertin, J.D. Tenhunen, R. Steinbrecher and G. Seufert. (2002). Monoterpene emissions in relation to foliar photosynthetic and structural variables in Mediterranean evergreen Quercus species. New Phytol. 153:243-256.
  33. Niinemets, Ü., G. Seufert, R. Steinbrecher and J.D. Tenhunen. (2002). A model coupling foliar monoterpene emissions to leaf photosynthetic characteristics in Mediterranean evergreen Quercus species. New Phytol. 153:257-276.
  34. Niinemets, Ü. (2002). Stomatal conductance alone does not explain the decline in foliar photosynthetic rates with increasing tree age and size in Picea abies and Pinus sylvestris. Tree Physiol. 22:515-535.
  35. Aasamaa, K., A. Sôber, W. Hartung and Ü. Niinemets. 2002. Rate of stomatal opening, shoot hydraulic conductance and photosynthesis characteristics in relation to leaf abscisic acid concentration in six temperate deciduous trees. Tree Physiol. 22:267-276.
  36. Niinemets, Ü., A. Cescatti, A. Lukjanova, M. Tobias and L. Truus. 2002. Modification of light-acclimation of Pinus sylvestris shoot architecture by site fertility. Agric. For. Meteorol. 111:121-140.
  37. Niinemets, Ü., A. Portsmuth and L. Truus. 2002. Leaf structure and photosynthetic characteristics, and biomass allocation to foliage in relation to foliar nitrogen content and tree size in three Betula species. Ann. Bot. 89:191-204.
  38. Niinemets, Ü., D.S. Ellsworth, A. Lukjanova and M. Tobias. 2002. Dependence of needle architecture and chemical composition on canopy light availability in three North American Pinus species with contrasting needle length. Tree Physiol. 22:747-761.

    07.07.03

Agu Laisk

Head of the project