Flash-induced Thermoluminescence Research Articles

Interaction of N,N,N′,N′-tetramethyl- p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the Q B niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl- p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S 2Q B − and S 3Q B − (B-band), S 2Q A − (Q-band), and Y D +Q A − (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot–Kok model of the S-state transitions and binary oscillations of Q B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q B-site and to reduce the higher S-states of the Mn cluster.

The role of D1-Ala344 in charge stabilization and recombination in Photosystem II

The Ala344 residue of the D1 protein has been identified as a crucial residue of the catalytic cluster of the water-oxidizing complex, however, its function has not been fully clarified. Here we have used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-Ala344stop mutation on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutant cannot grow photoautotrophically it shows flash-induced thermoluminescence and chlorophyll fluorescence signals reflecting the stabilization of negative and positive charges on the Q(A) and Q(B) quinone electron acceptors, and stable Photosystem II donors, respectively. Decay of flash induced chlorophyll fluorescence yield is multiphasic in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with 6 ms, 350 ms, and 26 s time constants. When cells are illuminated with repetitive flashes, fired at 1 ms intervals, the 6 ms phase is gradually decreased with the concomitant increase of the 350 ms phase. After 45 min dark adaptation of mutant cells the 6 ms and 350 ms phases were significantly decreased and a very slow decaying component was formed. Flash induced oscillation of the thermoluminescence B band, which reflects the redox cycling of the water-oxidizing complex in the wild-type cells, was completely abolished in the D1-Ala344stop mutant. The results demonstrate that low efficiency photooxidation of Mn occurs in about 60% of the PSII centers. The photooxidizable Mn is unstable in the dark, and formation of higher S states is inhibited. In addition, the Q(A) to Q(B) electron transfer step is slowed down as an indirect consequence of the donor side modification. Our data indicate that the stabilization of a Mn ion by the alpha-carboxylate chain of the D1-Ala344 residue might represent one of the final steps in the assembly of functional catalytic sites for water oxidation.

The function of D1-H332 in Photosystem II electron transport studied by thermoluminescence and chlorophyll fluorescence in site-directed mutants of Synechocystis 6803.

The His332 residue of the D1 protein has been identified as the likely ligand of the catalytic Mn ions in the water oxidizing complex (Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. (2004) Science 303, 1831-1838). However, its function has not been fully clarified. Here we used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-H333E, D1-H332D and D1-H332S mutations on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutants are not photoautotrophic they all show flash-induced thermoluminescence and chlorophyll fluorescence, which originate from the S(2)Q(A) (-) and S(2)Q(B) (-) recombinations demonstrating that charge stabilization takes place in the water oxidizing complex. However, the conversion of S(2) to higher S states is inhibited and the energetic stability of the S(2)Q(A) (-) charge pair is increased by 75, 50 and 7 mV in the D1-H332D, D1-H332E and D1-H332S mutants, respectively. This is most probably caused by a decrease of E(m)(S(2)/S(1)). Concomitantly, the rate of electron donation from Mn to Tyr-Z(b) during the S(1) to S(2) transition is slowed down, relative to the wild type, 350- and 60-fold in the D1-H332E and D1-H332D mutants, respectively, but remains essentially unaffected in D1-H332S. A further effect of the D1-H332E and D1-H332D mutations is the retardation of the Q(A) to Q(B) electron transfer step as an indirect consequence of the donor side modification. Our data show that although the His residue in the D1-332 position can be substituted by other metal binding residues for binding photo-oxidisable Mn it is required for controlling the functional redox energetics of the Mn cluster.

Response of chlorina barley mutants to heat stress under low and high light.

Barley plants (Hordeum vulgare L.) of wild type and two chlorina mutants, chlorina 126 and chlorina f2, were subjected to 42°C for 5 h at light intensities of 100 and 1000 μmol photons m-2 s-1. The exposure of plants to heat stress at a light intensity of 100 μmol m-2 s-1 induced enormous proline accumulation, indicating that the effect of heat stress was stronger when it was combined with low light intensity. The functional activity of PSII, O2evolution and flash-induced thermoluminescence B-band amplitude were strongly reduced when plants were exposed to heat at low light intensity. The results clearly showed that high light intensity had a protective effect on photosynthetic activity when barley plants were treated with high temperature. Comparison of the thermosensitivity of wild type plants and chlorina mutants revealed that O2 evolution in chlorina 126 and, especially, in chlorina f2 was more sensitive to heat than in wild type.

The role of transmembrane electrochemical potential and phosphorylation of PS II proteins in temperature induced light emission from ATP-treated lettuce thylakoids

Changes in characteristics of flash-induced thermoluminescence (TL) glow curves in thylakoids of lettuce following incubation of the organelles with ATP, under illumination or in the dark, were investigated. TL bands were induced by 1 or 2 flashes fired at -10°C or 1°C in thylakoids: TL curves in control thylakoids which were dark-adapted or submitted to an illumination without ATP, can be deconvoluted as the sum of one single B band and minor contributions. In thylakoids incubated for 90 s with 0.5 mM ATP, either under light or in the dark (after a 90 s preillumination), bands presented complex shapes; after deconvolution, they appeared composed of a B band with a low Ea (activation energy): 0.6 e.v. as compared to 0.75 in control, and a supplementary band peaking at about 10°C. The band at low temperature was suppressed by low concentrations (10-20 nM) of valinomycin, nigericin or FCCP as well as by 10 mM ammonium chloride, leaving B bands with the same characteristics as in control material. Finally with higher nigericin concentrations, the bands became single B bands with high Ea (0.9 e.v.). These characteristics would define 3 different energized states (in the form of a transmembrane electrochemical potential) for thylakoids based upon the presence of the 10°C band and the value of the activation energy for the B band component. The presence of a large 10°C band was also correlated to the existence of a larger transmembrane pH gradient, in the dark, after an ATP-treatment, than in controls. The 10°C band was specifically suppressed by the action of low concentrations of alkaline phosphatase with minor changes in characteristics of the remaining B band suggesting that phosphorylation of PS II proteins is also involved in the appearance of this low temperature band. The main mechanism at the origin of the low temperature band would be a destabilization of S2/3QB (-) charge pairs in energized membranes.

Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40°C in wild-type thylakoids to about 30°C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak temperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similiar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of Q B. (e) Mutations at the Q B site not only influence the binding of Q B and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S 2 and S 3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the Q B site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and Q B, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect Q B or DCMU binding in Synechococcus PCC7942.

Thermoluminescence and flash-oxygen characterization of the IC2 deletion mutant of Synechocystis sp. PCC 6803 lacking the Photosystem II 33 kDa protein

The psbO gene product of Photosystem II (PS II), the so-called 33 kDa extrinsic protein, is believed to be closely associated with the catalytic Mn cluster responsible for light-induced water oxidation. However, this protein is not absolutely required for water-splitting and its precise role remains to be clarified. We have used flash-induced thermoluminescence and oxygen evolution measurements to characterize the process of water oxidation in the IC2 mutant of Synechocystis sp. PCC 6803 from which the psbO gene had been deleted by Mayes et al. (Mayes, S.R., Cook, K.M., Self, S.J., Zhang, Z. and Barber J, (1991) Biochim. Biophys. Acta 1060, 1–12). The thermoluminescence results show that the extent of charge stabilization in the S 2Q A and S 2Q B states is reduced in the IC2 mutant to about 25–30% of that observed in the wild-type, suggesting that functional oxygen evolution occurs in a proportion of the psbO-less mutant cells. The stability of the S 2Q A, but not that of the S 2Q B, charge pair is markedly increased in the mutant. This points to a structural change of the PS II reaction center complex in the absence of the psbO gene product which affects the redox properties of the Q A and Q B acceptors to a different extent. The flash-induced oscillation of the B thermoluminescence band, arising from the S 2Q B and S 3Q B charge recombinations, is largely dampened in the mutant. This indicates that the ability of the water-oxidizing complex to reach its higher oxidation states, S 3 and S 4, is limited when the psbO gene product is absent. In agreement with the thermoluminescence results, flash-induced oxygen evolution shows a decreased yield and largely dampened oscillation pattern in the mutant. These results indicate that although the psbO gene product is not an absolute requirement for water oxidation its absence disturbs the redox cycling of the water-oxidizing complex and retards the formation of its higher S states. The rapid loss of thermoluminescence intensity during strong illumination of the mutated organism confirms its high susceptibility to photoinhibition. This effect is most likely the consequence of the limited rate of electron donation from the psbO-less water-oxidizing complex to the PS II reaction centre where the accumulation of highly oxidizing species may damage their pigment and protein surroundings.

Thermoluminescence and flash-oxygen characterization of the IC2 deletion mutant of Synechocystis sp. PCC 6803 lacking the Photosystem II 33 kDa protein

The psbO gene product of Photosystem II (PS II), the so-called 33 kDa extrinsic protein, is believed to be closely associated with the catalytic Mn cluster responsible for light-induced water oxidation. However, this protein is not absolutely required for water-splitting and its precise role remains to be clarified. We have used flash-induced thermoluminescence and oxygen evolution measurements to characterize the process of water oxidation in the IC2 mutant of Synechocystis sp. PCC 6803 from which the psbO gene had been deleted by Mayes et al. (Mayes, S.R., Cook, K.M., Self, S.J., Zhang, Z. and Barber J, (1991) Biochim. Biophys. Acta 1060, 1–12). The thermoluminescence results show that the extent of charge stabilization in the S 2 Q A and S 2 Q B states is reduced in the IC2 mutant to about 25–30% of that observed in the wild-type, suggesting that functional oxygen evolution occurs in a proportion of the psbO -less mutant cells. The stability of the S 2 Q A , but not that of the S 2 Q B , charge pair is markedly increased in the mutant. This points to a structural change of the PS II reaction center complex in the absence of the psbO gene product which affects the redox properties of the Q A and Q B acceptors to a different extent. The flash-induced oscillation of the B thermoluminescence band, arising from the S 2 Q B and S 3 Q B charge recombinations, is largely dampened in the mutant. This indicates that the ability of the water-oxidizing complex to reach its higher oxidation states, S 3 and S 4 , is limited when the psbO gene product is absent. In agreement with the thermoluminescence results, flash-induced oxygen evolution shows a decreased yield and largely dampened oscillation pattern in the mutant. These results indicate that although the psbO gene product is not an absolute requirement for water oxidation its absence disturbs the redox cycling of the water-oxidizing complex and retards the formation of its higher S states. The rapid loss of thermoluminescence intensity during strong illumination of the mutated organism confirms its high susceptibility to photoinhibition. This effect is most likely the consequence of the limited rate of electron donation from the psbO -less water-oxidizing complex to the PS II reaction centre where the accumulation of highly oxidizing species may damage their pigment and protein surroundings.

An Application of Thermoluminescence to Herbicide Studies

Thylakoids of genetically engineered species of Synechococcus PCC7942 were investigated by thermoluminescence (TL), polarographic and herbicide-binding studies. The results were as follows: 1) Deletion of either copy I or copy II/III of the psbA gene family resulted in modified oscillation patterns of flash induced TL and O2 evolution compared to wild type cells. 2) Replacement of Ser264 in psbA I by Ala or Gly inducing strong herbicide resistance leads to a downshift of the TL peak temperature of B-band by 10 C and alters the oscillation pattern of flash induced TL and O2 evolution significantly to exhibit a pattern with comparatively high O2 yield after the 2nd flash. Additional deletion of psbA II III does not lead to significant changes compared to the single mutated strains. 3) Contrary to Ser,M mutants, replacement of Phe255 by Tyr in psbA I alone, which also induces herbicide resistance, is not accompanied by- corresponding changes in TL peak temperature and/or oscillation pattern of flash-induced TL and O2 evolution. However, strongly altered properties are observed after additional deletion of psbA II/III. Based on these data it is inferred that expression of either psbA I or psbA II III genes only gives rise to PS II complexes slightly different in their function. Site-directed muta- genesis of psbA I gene modulates the expression ratio between copies I and II/III. The possible implications are discussed.

Modified Photosystem II acceptor side properties upon replacement of the quinone at the Q B site with 2,5-dimethyl- p-benzoquinone and phenyl- p-benzoquinone

By using a simple heptane/isobutanol extraction procedure on aqueous suspensions of thylakoids, the secondary quinone acceptor molecule at the Q B intermediate in Photosystem II was selectively removed and replaced with simple benzoquinone molecules, without significantly disturbing the primary quinone acceptor molecule at the Q A intermediate or the oxidizing side of the Photosystem II reaction center. The functioning of these modified samples was monitored by thermoluminescence measurements and the following observations were made. The extracted thylakoids, without any additions, exhibited a flash-induced thermoluminescence band identical with a DCMU-generated band (arising from S 2Q − A charge recombination) rather than a normal B band (arising from S 2Q − B charge recombination). The band did not display period four behavior in intensity following excitation by a sequence of light flashes. Upon the addition of either 2,5-dimethyl- p-benzoquinone or phenyl- p-benzoquinone to the extracted thylakoids, new thermoluminescence bands were generated. These new bands exhibited peak-temperature positions, intensities and room temperature decay kinetics that were distinctly different from each other as well as from the DCMU-generated band and the normal B band. However, the benzoquinone-generated bands remained sensitive to DCMU addition and did display period four behavior in intensity in a sequence of light flashes. These observations, therefore, indicate that the functioning of the Q B intermediate in Photosystem II can be reactivated in the extracted thylakoids by the addition of simple benzoquinones, although with modified properties. For comparison with the above results, data are also reported on heptane/isobutanol-extracted lyophilized thylakoids which were subsequently reconstituted with native plastoquinone.

Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks

A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S 2Q − A state while that at 25°C is attributed to recombination of S 2Q AQ − B (and S 3Q AQ − B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S 2Q AQ − B and S 3Q AQ − B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to Q A and thence to Q B or Q − B) results in predictable changes in the extent of flash-induced thermoluminescence.