Lecture 21

Fluorescence in photosynthesis

Fluorescence from photosystem II

In green plants, the fluorescence under physiological conditions comes from the photosystem II linked antenna, and provides a useful means of assaying various physiological parameters which affect photosystem II function. The fluorescence reflects the competition between several pathways for the excitation captured in the antenna. Under low-light conditions when photochemistry occurs with its maximal efficiency, excitation is passed mainly to the photoreactions (with an effciency >90%). When the photochemical traps are closed, excitation is lost by a competition between fluorescence and non-radiative dissipative pathways, the latter converting the energy to heat.

Because the fluorescence yield varies inversely with the fraction of open reaction centers, it provides a useful tool for investigation of photosynthetic processes. The yield can be assayed either from the fluorescence during continuous illumination, or from the fluorescence yield of a weak (<1% actinic) measuring flash. The later approach can be used to measure the reopening of the reaction center after a strong actinic flash.

Fluorescence kinetics to assay the reduction of plastoquinone by photosystem II

On illumination by continuous light, the fluorescence of a photosynthetic system rises from a minimal level (F0) by a complicated kinetics, which depends on the physiological state of the system.

The fluorescence yield is determined by a number of factors:

  1. Fluorescence changes associated with photochemical quenching (qQ-quenching) in open reaction centers. Reaction centers ‘close’ as the acceptor, QA, becomes reduced (either on reduction of the plastoquinone pool or because the photon flux exceeds the capacity for reoxidation), and the fluorescence rises.
  2. Centers can also become ‘closed’ through oxidation of the primary donor, but in this case fluorescence remains low because P680+ acts as a static quencher. It has generally been assumed in measurements with intact plants that this process is negligible, but this has not been adequately tested.
  3. The oxidized plastoquinone of the pool is a quencher, which has been largely ignored in most previous work, but has recently been shown to contribute significantly to the quenching under conditions where PSII efficiency is high.
  4. Formation of Chl triplets causes a quenching, which can be significant at high light intensities.
  5. Fluorescence lowering, or qE-quenching associated with the dumping of excess excitation energy under conditions of high light, which follows the generation and decay of low lumenal pH.
  6. Fluorescence lowering is modulated by formation of zeaxanthin and/or antheraxanthin, and may require that these de-epoxidation products of violaxanthin are present. The de-epoxidase of the lumen shows a strong dependence on pH, with increased activity as the pH falls below ~5.5.
  7. Irreversible quenching associated with photoinhibition (qI) which occurs when PS II is damaged, especially on the donor side.
  8. The redox state of the acceptor pools in intact systems reflects the state of activation of the assimilatory pathways, and also the effect of the back pressure from the proton motive force (back pmf) on the differential rate of filling and emptying of the quinone pool.
  9. The redox state of the donor side reactions is also strongly dependent on the lumenal pH, since protons are generated in the lumen is a product of water oxidation, and their activity enters directly into the mass action equation. Because the redox potentials of the partial reactions are not much lower than that of the P680/ P680+ couple at neutral pH, the equilibrium constants of the donor side are modulated so as to favor oxidation of P680 as the internal pH drops.
  10. State 1 - 2 transitions lead to changes in fluorescence associated with changes in absorption cross-section distribution between the photosystems.

Fluorescence induction

The yield of fluorescence (fF) during the initially phases (the induction of fluorescence) on continuous illumination, depends mainly on the state of the plastoquinone pool, and the competition between electron delivery to the pool, and exit from the pool. Delivery is determined by the light intensity, and the functionality of photosystem II. In a physiologically competent system illuminated at intensities in the ambient range, we can consider three conditions which exemplify useful experimental regimes.

  1. The system inhibited by DCMU. When DCMU is present in excess to inhibit reduction of the quinone pool through the QB-site of photosystem II, the fF rises from the F0 level to a maximal level (Fmax) as QA is reduced through the photochemistry of the reaction center. The rate depends on light intensity. The value of Fmax, DCMU depends on the state of the pool, since Q (but not QH2) is a weak quencher. Fmax can be as much as 30% higher with the pool reduced than with the pool oxidized. The area over the induction curve reflects the stoichiometry of the acceptor pool; with DCMU present, this is 1, because each photosystem II has one primary acceptor, QA.
  2. Electron exit from the quinone pool inhibited. This can be achieved by use of inhibitors of the bf-complex, by destruction of plastocyanin, or by excluding photosystem I acceptors. In these conditions, electrons fill the acceptor pool, and the fF rises to the Fmax level seen with DCMU when the pool is reduced. The area above the rise curve reflects the size of the acceptor pool, which can be normalized by comparison with the area above the curve in the presence of DCMU to get a value for the stoichiometry with respect to photosystem II.
  3. Electron acceptor present. When an acceptor of electrons from any point in the chain after photosystem II is present, the induction curve is modified by the competition between photochemical reduction and oxidation by acceptor. The shape of the induction curve, the maximal level of fluorescence, and the detailed kinetics, depend ina complicated fashion on the interplay between the factors listed above.

Kinetics of fF change on flash illumination

When measured in the range 0-10 ms following illumination by a short laser or xenon flash, the induction kinetics reflect the following reactions:

The half-times and fluorescence yield (fF) after a flash are shown below (t½ values and reactants vary with starting S-state (value for n) and the initial state of QB.):

                                    low fF                         high fF                         high fF                        low fF
Sn.Z.P.QA.QB ==> Sn.Z.P+.QA-.QB ==> Sn.Z+.P.QA-.QB ==> Sn+1.Z.P.QA-.QB ==> Sn+1.Z.P.QA.QB-

                       <1 ns                      20 - 200 ns                   30 µs - 1.5 ms            150-400 µs

The fluorescence rise kinetics over the time scale < 2 µs reflect reduction of P680+ (a quencher) (eq. 4),

Sn.Z.P+ <===> Sn.Z+.P       {4}

Sn.Z+.P <===> S(n+1)+.Z.P  {5}

A phase with t½ = ~ 30 - 400 µs reflects reduction of the oxidized donor tyrosine (YZ+, shown as Z+ in the equations) by the S-states and the displacement of the equilibration between states Z+.P and Z.P+ (eq. 5). In the absence of DCMU, this phase is convoluted with the contributions from oxidation of QA- (see below).

Longer lived low fluorescence states show inhibition on the donor side. By exploring the flash number dependence of these effects, the site of inhibition on the donor side can be located. Because the half-times on acceptor and donor sides vary with flash number, the binary and quartenary patterns associated with the two-electron gate and the OEC respectively, can be measured by appropriate choice of time after the actinic flash, and used to assay these reactions.

Kinetics of the two-electron gate

Kinetics in the range 30 µs - 2 ms reflect the oxidation of QA-. The kinetics depend on the state of QB, and are more rapid when QB is oxidized then when it is in the semiquinone state (QB-) before the flash. When the pool quinone and QB areoxidized, the QB-site can be either occupied or vacant, giving mixed first-order and pseudo-first order kinetics following a flash from the dark-adapted state. These differential kinetics can be used to explore the binary pattern of the two-electron gate, and the kinetic and thermodynamic parameters which describe its mechanism.

Nonphotochemical quenching of fluorescence

Plants protect themselves against excess light by switching on additional pathways for dissipation ("exciton dumping"), through a mechanism which leads to a lowering of fluorescence, and is general called non-photochemical quenching of fluorescence, or qE-quenching (the subscript E refers to the dependence on the state of "energization" of the chloroplast membrane by the proton gradient). This dissipative pathway requires two conditions, both of which depend on the lumenal pH:

  1. When lumenal pH falls below ~5.5, the enzyme violaxanthin de-epoxidase is activated, and converts violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle, mainly in the minor ligh harvesting complexes (LHCs), CP24, CP26 and CP29.
  2. When antheraxanthin and zeaxanthin are present in the minor LHCs, a quenching state is produced by low lumenal pH (<5.5) which probably reflects a change in the interaction between chlorophylls and the xanthophylls leading to thermal efficient dissipation through energy transfer.
  3. A brief review of the mechanism of qE-quenching can be found here.

©Copyright 1996, Antony Crofts, University of Illinois at Urbana-Champaign, a-crofts@uiuc.edu