JXB Advance Access originally published online on August 13, 2004
Journal of Experimental Botany 2005 56(411):337-346; doi:10.1093/jxb/erh237
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RESEARCH PAPER |
Singlet oxygen production in photosynthesis
Institut für Biologie II, Biochemie der Pflanzen, Universität Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany
* Fax: +49 761 203 2601. E-mail: anja.liszkay{at}biologie.uni-freiburg.de
Received 21 April 2004; Accepted 25 June 2004
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Abstract |
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Top
Abstract
Singlet oxygenA photosynthetic organism is subjected to photo-oxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by ß-carotene,
-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress. Key words: Light energy, photo-oxidative stress, photosynthesis, photosystem II, singlet oxygen
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Singlet oxygen |
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Living in an oxygen-rich world carries the potential risk of oxidative stress. Oxygen in the ground state is not directly a problem because it is relatively stable compared with its intermediates (peroxide (H2O2), superoxide
and hydroxyl radicals (OH·)). The relatively stable ground state of oxygen is a triplet state with two unpaired electrons with the same spin quantum number, each located in different antibonding (
*) orbitals. Oxygen can react by oxidizing another molecule, but, despite its high thermodynamic reactivity, its reactions are kinetically slow because of the spin restriction. Electron transfer reactions in the presence of oxygen can give rise to the production of the reactive intermediates, which themselves can produce different kinds of damage in the cell (Halliwell and Gutteridge, 1998
).
In addition, the very reactive singlet oxygen can be generated by an input of energy. In this state, the spin restriction is removed and therefore the oxidizing ability of the oxygen is greatly increased. Singlet oxygen is produced by light absorption by photosensitizers and, in plants, particularly by the chlorophylls and their precursors. On the one hand chlorophylls are needed for the use of light energy in photosynthesis, on the other hand, the same molecules carry the potential danger of being a singlet oxygen producer (photosensitizer). 1O2 has a short half-time of about 200 ns in cells (Gorman and Rodgers, 1992), and reacts with target molecules in the immediate neighbourhood. The possible diffusion distance of 1O2 has been calculated to be up to 10 nm in a physiologically relevant situation (Sies and Menck, 1992).
In the following, the reactions leading to the production of 1O2 in the antenna and reaction centres of the photosynthetic apparatus, the potential target molecules and the protection mechanism avoiding 1O2 production, are described (Fig. 1). In addition, the ‘useful’ role of 1O2 will be discussed, being not only a damaging species but also an element of signal transduction chains leading to the specific expression of stress-related genes.
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Special properties of chlorophyll |
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Chlorophyll as the main light-absorbing pigment in the light-harvesting complex, the inner antenna, and also in the reaction centres, is very efficient in absorbing light and has the additional advantage that the excited states are long-lived enough (up to a few nanoseconds) to allow the conversion of the excitation energy into an electrochemical potential via charge separation. If the energy is not efficiently used, the spins of the electrons in the excited state can rephase and give rise to a lower energy excited state: the chlorophyll triplet state. The chlorophyll triplet state has an even longer lifetime (a few µs under O2-saturated conditions) and can react with 3O2 to produce the very reactive 1O2 if no efficient quenchers are around. Chl triplet states may be populated in principle either directly by intersystem crossing (changing of the spin) from a singlet excited chlorophyll, or by charge recombination reactions (reversal of the charge separation and electron transfer reactions) in the reaction centres.
1O2 formation is favoured under certain physiological conditions like exposure to high light intensities or drought, leading to closure of the stomata and low CO2 concentrations in the chloroplasts. Under such conditions the plastoquinone pool can be in a very reduced state, forward electron transport is very limited, and recombination reactions in PSII can occur. The kinetically limiting step of the photosynthetic electron transport chain is thought to be the quinol oxidation in the Qo site of the cytochrome b6f complex.
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Quenching of chl triplet states and photoinhibition of PSII |
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1O2 can react with proteins, pigments, and lipids and is thought to be the most important species responsible for light-induced loss of PSII activity, the degradation of the D1 protein (protein of the reaction centre of PSII) and for pigment bleaching (for reviews on photoinhibition, see Prasil et al., 1992
; Aro et al., 1993). 1O2 formation in vivo was measured in the leaves of Arabidopsis thaliana by the use of a fluorescent dye (Hideg et al., 2001; op den Camp et al., 2003). Trebst and coworkers (Trebst et al., 2002) provided evidence that 1O2 is the important damaging species during photoinhibition (i.e. the light-induced loss of PSII activity and of the D1 protein) of Chlamydomonas reinhardtii cells.
The dangerous triplet state of chlorophylls, which is the origin of the observed 1O2, can be quenched directly by carotenoids in close proximity. The edge-to-edge distance between the two molecules must be less than the van der Waals distance (3.6 Å), i.e. the electron orbitals must have some overlap. In this spin exchange reaction, the triplet state of the carotenoid is formed which can either dissipate the excess energy directly as heat or by physical quenching via enhanced intersystem crossing with 3O2 (Edge and Truscott, 1999). This possibility is given in the antenna system, but not in the reaction centre, although two ß-carotene molecules are present in the PSII reaction centre (Telfer, 2002; for the location of the carotenes in the reaction centre, see Kamiya and Shen, 2003; Ferreira et al., 2004). In the reaction centre, the distance between the carotenes and the triplet chlorophyll is too large to allow a direct triplet quenching. The redox potential of the redox couple 
is very positive and a too close contact to the carotene would lead to the efficient oxidation of the carotene. Hence the primary function of these ß-carotenes is probably the quenching of 1O2 produced via the triplet state of P680 (Telfer, 2002). The latter was generated by charge recombination in PSII of the primary pair, P680Pheo. 1O2 can react with carotenoids which act as a catalyst, deactivating 1O2.
Another important antioxidant located in the thylakoid membrane is -tocopherol. Tocopherol is an efficient scavenger, which becomes oxidized when reacting with 1O2 (Trebst, 2003). Trebst et al. (2002) showed that inhibition of tocopherol biosynthesis in Chlamydomonas resulted in a stimulation of light-induced loss of PSII activity and D1 protein degradation. This implies that tocopherol comes close to the site of 1O2 generation in the reaction centre of PSII.
If 1O2 produced via chl triplet formation in the reaction centre is not quenched by carotenoids or tocopherol, it is very probable that it reacts with the D1 protein as a target molecule. The rapid turnover of the D1 protein occurs even at low light intensities (Keren et al., 1995), indicating that there is always some 1O2 formation even under low or moderate illumination. Singlet-oxygen-generating chemicals produce the same specific fragments of the D1 protein as are found under the conditions of acceptor side photoinhibition (Okada et al., 1996). The degradation of the D1 protein may be regarded as a physiological defence system to prevent uncontrolled damage of PSII. The controlled destruction of the D1 protein seems to be an attractive safety valve to detoxify 1O2 directly at the place of its generation (Trebst, 2003). Damaged D1 protein is degraded and PSII is repaired efficiently by the assembly of newly synthesized D1 in the so-called D1 protein damage–repair cycle (for reviews see Prasil et al., 1992; Aro et al., 1993).
With respect to photoinhibition studies, one has to differentiate between two experimental conditions chosen in the laboratory for in vitro studies: the so-called acceptor side photoinhibition which is related to the formation of 3P680 by charge recombination in PSII, followed by 1O2 production and the so-called donor side photoinhibition which occurs in PSII with a non-functional or absent water-splitting complex. In this state, photoinhibition is caused by the accumulation of highly oxidizing species like 
(Jegerschöld et al., 1990; Blubaugh et al., 1991) and is not related to 1O2 production (Krieger et al., 1998).
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Triplet chl formation by charge recombination in the reaction centre of PSII |
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In the reaction centre of PSII, the first detectable radical pair formed after excitation by light is
with P680 being the primary electron donor and pheophytin the primary electron acceptor (for a recent review on PSII see Goussias et al., 2002; for the X-ray structure of PSII, see Zouni et al., 2001; Kamiya and Shen, 2003; Ferreira et al., 2004). In isolated PSII reaction centres which lack QA and a functional donor side, the primary charge pair 
recombines and a high yield of P680 triplet is formed (Durrant et al., 1990). In the absence of oxygen, the lifetime of the triplet state is approximately 1 ms and shortens in the presence of O2 to approximately 30 µs. This effect, and the decrease in the stability of the reaction centre and bleaching of chl, were considered to be indirect evidence for 1O2 formation (Durrant et al., 1990). 1O2 formation was detected directly by its luminescence (Macpherson et al., 1993) and by EPR spin trapping (Hideg et al., 1994).
In isolated functional reaction centres possessing a complete and fuctional donor and acceptor side, the next step of electron transfer after the formation of the primary radical pair 
leads to the reduction of the primary quinone acceptor QA. Subsequently, 
is reduced by electron donation from the redox active tyrosine TyrZ, which itself obtains an electron from the water oxidizing complex. Forward electron transfer is much faster than charge recombination reactions. However, charge recombination reactions can occur when the forward electron transport cannot proceed. If the primary quinone acceptor stays reduced because of a block of the forward electron transport (the so-called closed state of the reaction centre), the yield of the primary charge separation is lowered. It has been proposed that the presence of the semiquinone anion 
in closed PSII may raise the energy of the primary pair by an electrostatic interaction so that the driving force of the primary charge separation is decreased compared with open reaction centres (state of the centre with the oxidized quinone, QA) (van Gorkom, 1985; Schatz et al., 1988). In the closed reaction centre, however, if primary charge separation occurs, it is followed by recombination of the charges. Charge recombination in the primary pair will produce either the singlet ground state of P680 or the triplet state of P680. The triplet state in the reaction centre is not localized directly on P680, i.e. the chlorophyll thought to bear the positive charge, but delocalized to another monomeric chlorophyll which is tilted by 30° compared with P680 (van Mieghem et al., 1991; Kamlowski et al., 1996). In the presence of a large antenna (PSII in vivo), the yield of the primary pair formed in the presence of will be low (van Mieghem et al., 1995).
Under reducing conditions, i.e. in the presence of dithionite and light (van Mieghem et al., 1989) or anaerobiosis and light (Vass et al., 1992), QA becomes doubly reduced, thereby releasing the negative electrostatic effect on the energy of the primary pair, and a high yield of charge separation, recombination, and P680 triplet formation is observed (van Mieghem et al., 1989). The double reduction of QA and the high yield of 3P680 formation in such centres have been suggested to have some relevance to photoinhibition (van Mieghem et al., 1989; Vass et al., 1992). The occurrence of double reduced QA, however, has never been shown to occur under physiologically relevant conditions.
At cryogenic temperatures (around 20 K), different from the situation at ambient temperature, the primary radical pair is formed with a high yield, irrespective of the redox state of QA and the yield of the triplet state 3P680 is high both with and QAH2 (van Mieghem et al., 1995). The triplet decay is much faster with than with QAH2 present. At room temperature, the yield of the primary pair is reduced in the presence of 
but nevertheless, a significant yield of the primary pair is found in core complexes of Synechococcus (about half of that found in reaction centres with double reduced QA) (Schlodder and Brettel, 1988; van Mieghem et al., 1995). Significant amounts of singlet oxygen are produced in PSII with a large antenna under continuous illumination (Hideg et al., 1994; Fufezan et al., 2002), and they are most likely linked to chl triplet formation by charge recombination of the primary pair in PSII and not to chl triplet formation in the antenna. The production of singlet oxygen via chl triplet formation by charge recombination reaction is shown by the following experiments: (i) the induction of photoinhibition by repetitive single turnover flashes and (ii) the effect of the mid-point potential of the redox couple 
on the yield of singlet oxygen production.
Photoinhibition of PSII by repetitive flashes
If dark-adapted PSII is excited by one single turnover flash (a short saturating flash which leads to one charge separation event in the majority of reaction centres), the state 
is formed, with S2 being an oxidation state of the Mn cluster, the water oxidizing complex, and QB, the secondary quinone acceptor. In the dark, the charges recombine via the formation of the primary radical. As already described above, charge recombination of the primary radical pair leads to the production of the singlet and the triplet state of P680. The chl triplet state can react with 3O2 leading to the formation of 1O2 which will damage the reaction centre. This flash-induced charge recombination reaction was exploited to investigate the mechanism of photoinhibition under low light in vivo (Keren et al., 1995) and in vitro (Keren et al., 1997). Keren and coworkers used a series of single turnover flashes, spaced with a dark interval of 32 s, and measured the degree of photoinactivation and loss of the D1 protein. The dark interval is long enough to allow charge recombination between the S2 or S3 state and 
to occur (the half-time of recombination is approximately 20 s; Rutherford and Inoue, 1984). When they used groups of flashes with 0.1 s spacing between the flashes in one group (for example, two flashes with 0.1 s interval and then 32 s dark interval), they observed photoinhibition after an uneven number of flashes per group and no or little photoinhibition after illumination with an even number of flashes per group. An even number of flashes produced the state S3QBH2 which does not recombine, while after an uneven number of flashes, charge recombination between the S2 or S3 state and occurs leading to singlet and triplet P680. This shows that an overall smaller number of flashes (less light absorption in total) can be more damaging than a greater number of flashes. This study was extended using Ca2+-depleted PSII preparations which were not active in water-splitting (Keren et al., 2000). In Ca2+-depleted PSII, the Mn cluster is blocked in the S3 state (for a review on Ca2+-depleted PSII see Debus, 1992). In the single turnover flash experiments, the loss of PSII activity was measured and compared with active samples. No difference between groups of even and uneven numbers of flashes was seen in Ca2+-depleted material. Using an uneven number of flashes, the activity loss was much smaller than in active samples. The yield of primary charge separation was not significantly reduced in Ca2+-depleted PSII, even after several single turnover flashes (Keren et al., 2000), implying that a difference in the charge recombination pathway must be responsible for this phenomenon.
Influence of the redox potential of the quinone acceptor on the yield of singlet oxygen formation
In Ca2+- and also in Mn-depleted PSII not only the water-splitting activity is inhibited but, in addition, the mid-point potential of the redox couple is up-shifted by about 150 mV. In PSII with an active water-splitting complex, the mid-point potential of the couple was found to be –80 mV (Krieger and Weis, 1992; Krieger et al., 1995). In centres with the high potential form of QA (Em about +65 mV), forward electron flow from QA to QB is energetically disfavoured and electron transfer is therefore unlikely to occur (Fig. 2) (Johnson et al., 1995; Krieger et al., 1995; Andréasson et al., 1995). It was proposed that, in such centres, the shift of the mid-point potential of QA influences the pathway of charge recombination within the reaction centre of PSII. In active PSII, with QA in its normal, low potential form, charge recombination between the acceptor and the donor side proceeds with a high probability via the formation of the primary pair 
resulting in the formation of singlet and triplet P680. In centres with the high potential form of QA, the formation of the primary pair is not disfavoured (Keren et al., 2000) and charge recombination may occur via an alternative pathway which does not involve the formation of excited chlorophyll species (Fig. 3; see also Johnson et al., 1995; Rutherford and Krieger-Liszkay, 2001). As already described above, the loss of PSII activity after excitation by an uneven number of flashes was about 30% less in Ca2+-depleted PSII than in active PSII. In addition, no singlet oxygen production could be measured by spin trapping EPR with TEMP under continuous illumination (Krieger et al., 1998). This shows that the change of the mid-point potential of QA is an important molecular switch for changing the charge recombination pathway within PSII. By changing the mid-point potential of QA from low to high potential, the formation of singlet oxygen can be avoided.
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because the spins had time to randomize. The triplet state of this radical pair can recombine rapidly, resulting in 3P680. 3P680 can react with 3O2 forming 1O2. The influence of herbicide binding on the mid-point potential of the redox couple This regulation mechanism of PSII may be of physiological importance. PSII is assembled without the Mn cluster and with QA in the high potential form (Johnson et al., 1995
). In the light, during the so-called photoactivation process, the Mn cluster is assembled and the mid-point potential of QA is switched from high potential to the low potential form, which allows linear electron flow. In the state prior to complete assembly of the functional water-splitting complex, PSII is protected against photodamage induced by 1O2 formation. Under high light conditions, the change of the mid-point potential of QA may also be involved in the pH-dependent control of PSII activity. In addition to the dissipation of excess energy by the formation of zeaxanthin in the antenna (for a review see Demming-Adams, 1990), the activity of the electron transfer can be altered at the level of the reaction centre of PSII (reaction centre quenching). When, in excess light, the pH in the lumen decreases below a certain threshold value, up to one Ca2+ per PSII can be released and the mid-point potential of QA is thereby switched to the high potential form (Krieger and Weis, 1992). This was demonstrated in thylakoid membranes, in which a proton gradient was maintained by ATP-hydrolysis in the dark, by measuring the chlorophyll fluorescence at the Fo-level at different redox potentials as a measure for the reduction state of QA (Krieger and Weis, 1993).
It is still unclear how the activity state of the water-splitting complex at the donor side is connected to the mid-point potential of the quinone at the acceptor side of PSII. One possibility is that, upon the release of Ca2+, a structural change in a protein subunit of the reaction centre and especially at the QA binding site occurs, which could be responsible for the observed change in the mid-point potential. It might also be possible that cytochrome b559 mediates between the donor and acceptor sides. In inactive and non-photoactivated PSII, cytochrome b559 is in the low potential form and changes upon the assembly of the Mn to the high potential form, characteristic for the active PSII (for review, see Stewart and Brudvig, 1998). The change of the potential form of cytochrome b559 was already observed before the process of photoactivation was fully completed (Mizusawa et al., 1997).
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Influence of herbicides on the mid-point potential of QA and on singlet oxygen production |
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The influence of different herbicides on the mid-point potentials of the primary quinone acceptor QA (Krieger-Liszkay and Rutherford, 1998
) and single point mutations in D1 (Rappaport et al., 2002) or in D2 (Vavilin and Vermaas, 2000) can be used as a tool to investigate the charge recombination pathways in PSII. Binding of herbicides to the QB binding site of the D1 protein inhibits linear electron flow and affects the degree of photoinactivation and light-induced degradation of the D1 protein. In vitro, the urea herbicide DCMU and related herbicides have been reported to retard photodamage (Keren et al., 1995, 1997; Kirilovsky et al., 1994) and degradation of the D1 protein (Keren et al., 1995, 1997; Nakajima et al., 1996; Jansen et al., 1993; Zer and Ohad, 1995). By contrast with DCMU, phenolic herbicides, which also bind to the QB-binding site, have the opposite effect and stimulate the susceptibility of PSII to light (Pallett and Dodge, 1980; Nakajima et al., 1996) and the degradation of D1 (Jansen et al., 1993). Binding of these herbicides to the QB binding site influence the mid-point potential of QA. Phenolic herbicides lower the mid-point potential by approximately 45 mV and DCMU raises it by about 50 mV (Krieger-Liszkay and Rutherford, 1998). A smaller difference of 60 mV between the redox potential in the presence of bromoxynil and in the presence of DCMU was reported by Roberts et al. (2003) when estimated from the back reaction rate of 
The effect of the different types of herbicide on the mid-point potential of QA was not only observed for the low potential form but also for the high potential form of QA (Krieger-Liszkay and Rutherford, 1998). The absolute change in the mid-point potential of QA by these herbicides was much lower (±50 mV) than the shift induced by inactivation of the water-splitting complex (Ca2+- or Mn-depletion), but it has, nevertheless, a big effect on the yield of 1O2 production. The molecular basis for the shift of the mid-point potential of QA by binding a herbicide to the QB binding pocket is not understood. FTIR spectra of QA obtained in the presence of a phenolic herbicide compared with DCMU indicate that the protein environment of QA is slightly modified by the phenolic herbicide. The change seen in the spectra is small and approximately in the range of one H-bonding (J Breton and A Krieger-Liszkay, unpublished data).
Fufezan et al. (2002) showed that the yield of 1O2 production in the presence of a phenolic herbicide in active PSII-enriched membrane fragments (with QA in the low potential form) is twice as high as in the presence of DCMU. This effect is already seen at relatively low light intensities (400 µmol quanta m–2 s–1) and the amount of 1O2 produced increases linearly with increasing light intensities. In bacterial reaction centres it has been shown that the free energy gap between the 
radical pair and the P+BPheo– radical pair has a major influence on the back reaction pathway (Gunner et al., 1982; Gopher et al., 1985; Woodbury et al., 1986; Shopes and Wraight, 1987). When the gap is smaller than 400 meV, the back reaction via the primary pair (P+BPheo–) dominates, while under conditions where the gap is greater than this value, a direct recombination pathway dominates 
This direct recombination pathway involves electron tunnelling reactions. Based on these observations made with the bacterial reaction centre a model was proposed (Fig. 3) showing the influence of the mid-point potential of QA on the charge recombination pathway within PSII (Krieger-Liszkay and Rutherford, 1998; Rutherford and Krieger-Liszkay, 2001). It seems likely that the modulation of the mid-point potential of QA by the state of the water-splitting complex and by the herbicides will influence the free energy gap between 
and 
With DCMU it is predicted that the increase in the mid-point potential should increase the free energy gap and thereby diminish the yield of back reaction via the radical pair. By analogy to the bacterial reaction centre a direct recombination via may take place which does not result in the formation of excited singlet or triplet states of P680. This model may explain the lower production of 1O2 observed in the presence of DCMU in centres with low potential QA (Fufezan et al., 2002) and the absence of 1O2 formation in PSII with high potential QA (Krieger et al., 1998).
On the other hand, the decreased mid-point potential of QA induced by phenolic herbicides should make the energy gap between and smaller and, therefore, the back reaction via the primary radical pair and the formation of P680 triplet more likely.
Rappaport et al. (2002) investigated the influence of the mid-point potential of the Pheo/Pheo– redox couple on charge recombination between 
and showed that the recombination rate is sensitive to the free energy gap between Pheo and QA. They used mutants of Synechocystis, in which the mid-point potential of Pheo/Pheo– was shifted by +33 mV or –74 mV compared with the wild type. The mutant with the potential shift of +33 mV showed an increase in the recombination rate 
by a factor of four (measured as the decay of fluorescence after a saturating flash), while lowering of the mid-point potential slowed down the recombination by the same factor. This demonstrates that charge recombination via the formation of the primary radical pair is a significant process in PSII reaction centres in which forward electron flow is blocked. In addition they demonstrated that the direct charge recombination pathway which does not involve the repopulation of the primary pair is significant in the mutant with the lowered mid-point potential of Pheo/Pheo–. In a different set of experiments, mutations in the CD loop of the D2 protein were made in Synechocystis (Vavilin and Vermaas, 2000). These mutants show a temperature shift and a decrease in intensity in the thermoluminescence band originating from recombination. This was interpreted as an increase of the proportion of the direct recombination pathway of the pair which does not lead to the formation of an excited chlorophyll.
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Charge recombination and chl triplet formation in the reaction centre of PSI |
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In PSI, under reducing conditions when the iron sulphur clusters are prereduced or when vitamin K1 is removed from the reaction centre, charge recombination reactions also occur leading to the triplet state of P700 at room temperature (for a review see Brettel, 1997
). In PSI, the lifetime of the state 3P700 is about 6 µs and is not shortened by 3O2, indicating that P700 is screened from O2 (Sétif et al., 1981). |
1O2 production by the cytochrome b6f complex |
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Illumination of the isolated cytochrome b6f complex results in the formation of 1O2, as shown by spin trapping techniques and the effects of azide, a 1O2 quencher, and D2O, which extends the lifetime of 1O2 (Suh et al., 2000
). It was shown by Suh et al. (2000) that the Fe-S cluster of the Rieske protein and not the cytochromes are responsible for the 1O2 production in the light. The extent to which 1O2 generated by the cytochrome b6f complex contributes to photoinhibition of PSII is unclear. The cytochrome b6f complex contains, in addition to the other cofactors, one chlorophyll with an unknown function. In principle, this chlorophyll could also be involved in 1O2 formation acting as a photosensitizer. |
Chlorophyll triplet and singlet oxygen production in the antenna |
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Chlorophyll triplet states and consecutive 1O2 formation is not only produced by charge recombination in the reaction centre, but also by intersystem crossing from a singlet excited chlorophyll in the antenna. In addition, excited states of chlorophyll precursors can lead to the formation of 1O2.
In isolated protein/pigment complexes, the rate of intersystem crossing is significant (Kramer and Mathis, 1980) and the formation of triplet chlorophylls in the antenna has been shown in vitro. These states can be distinguished by their spectroscopic characteristics from the triplet chlorophyll in the reaction centre and they do not depend on the redox potential of the medium (Santabarbara et al., 2002). Two of these triplet states are probably generated in the core complex while the third one may be generated in the light-harvesting complex (Santabarbara et al., 2002). Formation of 1O2 from isolated LHCII has been shown in vitro by spin trapping with TEMP (Rinalducci et al., 2004). Singlet oxygen may also play a role in the degradation of light-harvesting proteins. The degradation of LHCII is much slower than the degradation of the D1 protein (Lindahl et al., 1995), but modifications of the protein are already visible after a few hours of low intensity illumination (100 µmol quanta m–2 s–1) in the isolated complex (Zolla and Rinalducci, 2002). Degradation of LHCII releases a large number of chl. Light-induced damage might occur from such chl which is energetically uncoupled from the antenna, and will give a high triplet chl and, therefore, possibly high 1O2 yield. However, experimental evidence for the production of 1O2 by triplet formation in the antenna and their involvement in the light-induced damage in PSII in vivo in mature leaves is still missing. In the antenna 3chl will be efficiently quenched by nearby carotenoids, so that 3chl, although formed with a higher probability than by charge recombination in the reaction centre, will rarely be a problem.
Production of triplet chlorophyll and 1O2 may play a role during the transition from etioplasts to chloroplasts. In greening material, disorganized chlorophyll may act as a photosensitizer (Marder et al., 1998). Oxygen uptake by thylakoid membranes, isolated from greening material, was measured. Oxygen uptake was significantly quenched by ß-carotene and -tocopherol, indicating that production of singlet oxygen was measured by this method (Caspi et al., 2000). Protochlorophyllide acts also as photosensitizer, as shown for the Arabidopsis mutant flu (op den Camp et al., 2003). In this mutant, a protein is inactivated which plays a key role during the negative feedback control of chlorophyll biosynthesis. As a consequence, the mutant accumulates free protochlorophyllide in the dark. In this study, the production of 1O2 was shown in leaves by quenching of the fluorescent dye DanePy in vivo after a dark–light transition of the plants (Hideg et al., 1998; op den Camp et al., 2003). It was also shown previously, by the use of herbicides which block the protoporphyrinogen oxidase, that protoporphyrin IX is a photodynamic pigment which produces high amounts of 1O2 in the light (Becerril and Duke, 1989).
1O2 may also be produced by free chlorophyll and chlorophyll degradation products which may be produced during strong photoinhibition. If the light-induced damage exceeds the controlled D1 degradation and repair of PSII, further protein degradation of chl binding subunits may lead to the production of free chls, which are dangerous photosensitizers. These free chls may be bound by ELIP (Early Light Induced Proteins) proteins (Adamska, 1997) or by proteins like WSCP (Water Soluble Chlorophyll Protein) (Schmidt et al., 2003). The binding of chl to the WSCP reduces the yield of 1O2 production by a yet unknown mechanism. This protein is an unusual chl-binding protein in the sense that it does not bind carotenoids, but, nevertheless, efficiently protects bound chl against photodegradation and reduces the yield of 1O2 production.
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Gene expression in response to 1O2 formation |
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There are recent reports in the literature that, as a response to 1O2 production, genes are specifically up-regulated which are involved in the molecular defence response of the plant against photo-oxidative stress (Leisinger et al., 2001
; op den Camp et al., 2003; B Fischer, personal communication). Leisinger et al. (2001) showed that, in the presence of photosensitizers like Rose Bengal, a glutathione peroxidase homologous gene from Chlamydomonas is transcriptionally up-regulated by 1O2, while the mRNA level of this gluthathione peroxidase is only weakly expressed by exposure to superoxide or peroxide. Op den Camp et al. (2003) used the flu mutant of Arabidopsis to show that the accumulation of protochlorophyllide and thus the high yield of 1O2 formation by transferring these plants from dark to light rapidly activated a number (70) of genes. By contrast, other reactive oxygen species like superoxide did not rapidly up-regulate the expression of these genes. In the flu mutant, 1O2 is produced peripherally at the membrane surface and can, therefore, react with compounds of the stroma. Under natural conditions, 1O2 will be produced within the reaction centre of PSII and will react with different target molecules than in this mutant. Fischer, however, using inhibitors of the photosynthetic electron transport, studied 1O2 formation in PSII and only found a significant up-regulation of the glutathione peroxidase homologous gene from Chlamydomonas (B Fischer, personal communication).
The question arises how an extremely short-lived molecule like 1O2 can give rise to a signal that can be transmitted to the nucleus to regulate gene expression. Some other reactive oxygen species like superoxide or peroxide have been shown to act directly as second messengers in the regulation of expression of the oxidative stress response genes such as gutathione peroxidases, glutathione-S-transferases, and ascorbate peroxidase (for reviews see Mullineaux et al., 2000; Vranová et al., 2002). Because of the short lifetime of 1O2, it can be excluded that it oxidizes a component of a signal transduction chain directly. Instead reaction products originating either from the D1 protein degradation or products of chlorophyll degradation can be envisaged as signal molecules. It has been shown that chlorophyll precursors like Mg-protoporphyrin IX can act as a signalling molecule in a signalling pathway between the chloroplasts and the nucleus (Strand et al., 2003). By analogy, one can also speculate that a chlorophyll degradation product such as pheophytin, chlorophyllide, or pheophorbide (for chl degradation, see Matile et al., 1999) may act as a signalling molecule. Such a molecule could be transported out of the chloroplast to the cytosol by an ABC protein where it mediates a signal to the nucleus to regulate the expression of genes. It has been shown that a functional ABC protein is required for the transport of protophorhyrin IX (Møller et al., 2001). It was also shown that an ABC transporter in the tonoplast membrane can transport chlorophyll catabolites to the vacuole (Lu et al., 1998), making it likely that such a transport mechanism is also present in the chloroplast envelope membrane.
Alternatively, lipid peroxides may function as signalling molecules because unsaturated fatty acids are the preferred targets of 1O2. However, no increase in 1O2-mediated non-enzymatic lipid peroxidation could be found in the flu mutant, which accumulates protochlorophyllide and shows a higher yield of 1O2 formation than wild-type plants (Op den Camp et al., 2003). Linolenic acid was rapidely oxidized upon illumination of the flu mutant, but the oxidation patterns observed were indicative for enzymatic oxidation and not for non-enzymatic oxidation by 1O2. In general, fatty acid-derived signals may be involved in signalling pathways connected with cell death and the expression of stress-related genes (Weber, 2002).
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Acknowledgements |
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I thank N Bondarava, B Fischer, C Fufezan, S Panahandeh, A Telfer, and A Trebst for stimulating discussions and critical reading of the manuscript.
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Footnotes |
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Abbreviations: chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; P680, primary electron donor in PSII; Pheo, pheophytin (primary electron acceptor in PSII); PS, photosystem, QA, primary quinone acceptor in PSII; QB, secondary quinone acceptor in PSII; TEMP, 2,2,6,6-tetramethylpiperidine-1-oxyl; TyrZ, redox active tyrosine of the D1 protein; S2, S3, oxidation states of the Mn cluster.
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