The quenching characteristics of potassium iridic chloride and their meaning for the origin of chlorophyll fluorescence components

To understand the origins of the different lifetime components of photosystem 2 (PS2) chlorophyll (Chl) fluorescence we have studied their susceptibility to potassium iridic chloride (K₂IrCl₆) which has been shown to bleach antenna pigments of photosynthetic bacteria (Loach et al. 1963). The addition of K₂IrCl₆ to PS2 particles gives rise to a preferential quenching of the variable Chl fluorescence (F_v). At concentrations lower than 20 M, this is brought about mainly by a decrease in the yield, but not in the lifetime, of the slowest component when all the PS2 reaction centres are closed (F_M). The yield of the middle and fast decays are not significantly altered. This type of quenching is not seen with DNB. The iridate-induced quenching of the initial fluorescence level (F₀) is due to a proportional decrease in the yield and lifetime of the three components and correlates with the observed modification in the relative quantum yield of oxygen evolution. In this concentration range a bleaching of Chl a is seen. At higher iridate levels, greater than 20 M, a proportional decrease in the lifetimes and yields of the three kinetic components is seen at F_M. These changes are associated with a carotenoid bleaching. In isolated light harvesting Chl a/b complexes of PS2 (LHC2), iridate addition converts a 4 ns decay into a 200 ps emission and both types of bleaching are observed. By also measuring the rate of PS2 trap closure versus iridate concentration, we have discussed the results in terms of excitation energy transfer.Abbreviations DNB m-dinitrobenzene - F_M maximum Chl fluorescence - F₀ initial fluorescence - F_v variable fluorescence - I pheophytin a primary electron acceptor of PS2 - P₆₈₀ chlorophyll a of photochemical centre - PS2 photosystem 2 - Q_A primary stable electron acceptor of PS2 - Chl chlorophyll - LHC2 light harvesting Chl a/b complex of PS2 - MES 2(N-morpholino) ethanesulfonic acid - DCMU 3-(3-4-dichlorophenyl) 1-1 dimethylurea - PPBQ phenyl-p-benzo-quinone - BBY PS2-enriched membranes prepared as in Berthold et al. (1981) - Q₄₀₀ PS2 electron acceptor with a midpoint potential of 400 mV     

Fluorescence characteristics of photoautotrophic soybean cells

We report here the first measurements on chlorophyll (Chl) a fluorescence characteristics of photoautotrophic soybean cells (cell lines SB-P and SBI-P). The cell fluorescence is free from severe distortion problems encountered in higher plant leaves. Chl a fluorescence spectra at 77 K show, after correction for the spectral sensitivity of the photomultiplier and the emission monochromator, peaks at 688, 696 and 745 nm, representing antenna systems of photosystem II-CP43 and CP47, and photosystem I, respectively. Calculations, based on the complementary area over the Chl a fluorescence induction curve, indicated a ratio of 6 of the mobile plastoquinone (including Q_B) to the primary stable electron acceptor, the bound plastoquinone Q_A. A ratio of one between the secondary stable electron acceptor, bound plastoquinone Q_B, and its reduced form Q_B ^- was obtained by using a double flash technique. Owing to this ratio, the flash number dependence of the Chl a fluorescence showed a distinct period of four, implying a close relationship to the S state of the oxygen evolution mechanism. Analysis of the Q_A ^- reoxidation kinetics showed (1) the halftime of each of the major decay components ( 300 s fast and 30 ms slow) increases with the increase of diuron and atrazine concentrations; and (2) the amplitudes of the fast and the slow components change in a complementary fashion, the fast component disappearing at high concentrations of the inhibitors. This implies that the inhibitors used are able to totally displace Q_B. In intact soybean cells, the relative amplitude of the 30 ms to 300 s component is higher (40:60) than that in spinach chloroplasts (30:70), implying a larger contribution of the centers with unbound Q_B. SB-P and SBI-P soybean cells display a slightly different sensitivity of Q_A ^- decay to inhibitors.Abbreviations CA complementary area over fluorescence induction curve - Chl chlorophyll, diuron - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F _m maximum chlorophyll a fluorescence - F ₀ minimum chlorophyll a fluorescence - F _v = F _t-F₀ - where F _v = variable chlorophyll a fluorescence - and F_t = chlorophyll a fluorescence at time t - PS II photosystem II - Q _a primary (plastoquinone) electron acceptor of PS II - Q _b secondary (plastoquinone) electron acceptor of PS II - t₅₀ the time at which the concentration of reduced Q _a is 50% of that at its maximum value     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q_A) of photosystem (PS) II [C. Vernotte, A.L. Etienne, J.-M. Briantais, Quenching of the system II chlorophyll fluorescence by the plastoquinone pool, Biochim. Biophys. Acta 545 (1979) 519-527]. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 μs to 5 s). The about 20% lowering of the maximum fluorescence yield F_M, observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH₂ by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH₂ starts getting reoxidised by PS I activity. NAD(P)H-dependent restoration of F_M was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl₂ that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F_M. Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F₀ allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F₀ level (Q₀) and to compare it with the fractional quenching at the F_M level (Q_M). The experimentally determined Q₀/Q_M ratios were found to be equal to the corresponding F₀/F_M ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Quantification of non-Q B -reducing centers in leaves using a far-red pre-illumination

An alternative approach to quantification of the contribution of non-Q_B-reducing centers to Chl a fluorescence induction curve is proposed. The experimental protocol consists of a far-red pre-illumination followed by a strong red pulse to determine the fluorescence rise kinetics. The far-red pre-illumination induces an increase in the initial fluorescence level (F_{25 μs}) that saturates at low light intensities indicating that no light intensity-dependent accumulation of Q_A⁻ occurs. Far-red light-dose response curves for the F_{25 μs}-increase give no indication of superimposed period-4 oscillations. F_{25 μs}-dark-adaptation kinetics following a far-red pre-pulse, reveal two components: a faster one with a half-time of a few seconds and a slower component with a half-time of around 100 s. The faster phase is due to the non-Q_B-reducing centers that re-open by recombination between Q_A⁻ and the S-states on the donor side. The slower phase is due to the recombination between Q_B⁻ and the donor side in active PS II reaction centers. The pre-illumination-induced increase of the F_{25 μs}-level represents about 4–5% of the variable fluorescence for pea leaves (∼2.5% equilibrium effect and 1.8–3.0% non-Q_B-reducing centers). For the other plant species tested these values were very similar. The implications of these values will be discussed.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Photochemical and Thermal Phases of Chlorophyll a Fluorescence

The measurement of variable chlorophyll (Chl) a fluorescence is widely used as a convenient and versatile tool in photosynthesis research. In many applications empirical correlations and simplified models of Chl a fluorescence are used with success. Nevertheless, variable Chl a fluorescence provides only indirect and complex image of processes occurring within photosynthetic membranes and such simplifications have only limited validity. In this review we elucidate some controversial and still unresolved questions about the origin and interpretation of the variable Chl a fluorescence induction and the proper use of variable Chl a fluorescence for studies of photochemical events in photosystem 2 (PS2). Although the major part of variable Chl a fluorescence reflects the photochemical closure of the PS2 reaction centers (RCs) and can be considered as a function of the redox state of the primary acceptor Q_A, up to 50 % of the change in the Chl a fluorescence yield can be of secondary, nonphotochemical origin. We review the possible sources of the inherent heterogeneity in the origin of variable Chl a fluorescence. We also comment on the practical implications this bears for the use of variable Chl a fluorescence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, F_o) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, F_m). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (pH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a pH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4–0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273–2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant pH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The pH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and pH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.Abbreviations Ax antheraxanthin - BSA bovine serum albumin - c_x lifetime center of fluorescence decay component x - CP chlorophyll binding protein of PS II inner antenna - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - f_x fractional intensity of fluorescence lifetime component x - F_m, F_m maximal PS II Chl a fluorescence intensity with all Q_A reduced in the absence, presence of thylakoid membrane energization - F_o minimal PS II Chl a fluorescence intensity with all Q_A oxidized - F_v=F_m–F_o variable level of PS II Chl a fluorescence - HPLC high performance liquid chromatography - k_A rate constant of all combined energy dissipation pathways in PS II except photochemistry and fluorescence - k_F rate constant of PS II Chl a fluorescence - LHCIIb main light harvesting pigment-protein complex (of PS II) - N_pig mols Chl a+b per PS II - NPQ=(F_m/F_m–1) nonphotochemical quenching of PS II Chl a fluorescence - PAM pulse-amplitude modulation fluorometer - PFD photon-flux density, mols photons m^–2 s^–1 - PS II Photosystem II - P680 special-pair Chls of PS II reaction center - Q_A primary quinone electron acceptor of PS II - V_x violaxanthin - w_x width at half maximum of Lorentzian fluorescence lifetime distribution x - Zx zeaxanthin - pH trans-thylakoid proton gradient - % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad2gaaeqaaaaa!4989!\[< \tau > _{Fm}\],% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad+gaaeqaaOGaeyypa0Zaaabqaeaaca% WGMbWaaSbaaSqaaiaadIhaaeqaaOGaam4yamaaBaaaleaacaWG4baa% beaaaeqabeqdcqGHris5aaaa!50D3!\[< \tau > _{Fo} = \sum {f_x c_x }\] average lifetime of Chl a fluorescence calculated from a multi-exponential model under F_m, F_o conditions     

Fractional control of photosynthesis by the QB protein,the cytochrome f/b 6 complex and other components of the photosynthesic apparatus

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the Q_B protein of the electron-transport chain. Fractional control by the cytochrome f/b ₆ complex was insignificant under these conditions. Control by the cytochrome f/b ₆ complex dominated at high energy fluence rates where the contribution to control of the Q_B protein was very small. Uncoupling shifted control from the cytochrome f/b ₆ complex to the Q_B protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b ₆ complex and of the Q_B protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO₂ saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b ₆ complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the Q_B protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2,4-dinitro phenylether of 2-iodo-4-nitrothymol - pCMBS p-chloromercuribenzosulfonate     

Dynamics of photosystem II heterogeneity in Dunaliella salina (green algae)

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from Q_A to plastoquinone (Q_B-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from Q_A to Q_B (Q_B-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of Q_B-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 E m^-2s^-1), dark incubation induces an increase (half-time 45 min) in the Q_B-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of Q_B-nonreducing centers to 25%. In cells grown under low illumination intensity (30 µE m^–2s^–1), dark incubation elicits no change in the relative concentration of Q_B-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the Q_B-nonreducing pool size and a concomitant increase in the Q_B-reducing pool size. These and other results are explained in terms of a pool of Q_B-nonreducing centers existing in a steady-state relationship with Q_B-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane of D. salina. It is proposed that Q_B-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the Q_B-nonreducing pool to maintain a constant pool size of Q_B-nonreducing centers in the thylakoid membrane.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F_o non-variable fluorescence yield - F_pl intermediate fluorescence yield plateau level - F_max maximum fluorescence yield - F_i mitial fluorescence yield increase from F_o to F_pl(F_pl-F_o) - F_v total variable fluorescence yield (F_max-F_o) - DCMU dichlorophenyl-dimethylurea     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y_D^ox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S₂Q_A⁻ and S₂Q_B⁻ charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y_D⁺Q_A⁻ recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S₁-to-S₂-transition and S₂-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q_A to Q_B electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

Bicarbonate effects in leaf discs from spinach

In this paper, we show the unique role of bicarbonate ion in stimulating the electron transfer of photosystem II (PS II) in formate-treated leaf discs from spinach. This is referred to as the bicarbonate effect and is independent of the role of CO₂ in CO₂ fixation. It is shown to have two sites of action:

Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q_A of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F _o towards a maximum F _m^STF when Q_A becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20–30% increase when the second partner is applied within 20–100 μs after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2–0.4 and 2–5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1–4 kHz) shows (i) in the first 3–4 flashes a transient overshoot 20–30% above the F _m^STF = 3*F _o level reached in the 1st flash with a partial decline towards a dip D in the next 2–3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F _m = 5*F _o in the 3–60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S₀ fraction with Q_B-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q_B-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.     

Analysis of chlorophyll a fluoresence changes in weak light in heat treated Amaranthus chloroplasts

After preheating of Amaranthus chloroplasts at elevated temperatures (up to 45°C), the chlorophyll a fluorescence level under low excitation light rises as compared to control (unheated) as observed earlier in other chloroplasts (Schreiber U and Armond PA (1978) Biochim Biophys Acta 502: 138–151). This elevation of heat induced fluorescence yield is quenched by addition of 0.1 mM potassium ferricyanide, suggesting that with mild heat stress the primary electron acceptor of photosystem II is more easily reduced than the unheated samples. Furthermore, the level of fluorescence attained after illumination of dithionite-treated samples is independent of preheating (up to 45°C). Thus, these experiments indicate that the heat induced rise of fluorescence level at low light can not be due to changes in the elevation in the true constant F₀ level, that must by definition, be independent of the concentration of Q_A. It is supposed that the increase in the fluorescence level by weak modulated light is either partly associated with dark reduction of Q_A due to exposure of chloroplasts to elevated temperature or due to temperature induced fluorescence rise in the so called inactive photosystem II centre where Q_A are not connected to plastoquinone pool. In the presence of dichlorophenyldimethylurea the fluorescence level triggered by weak modulated light increases at alkaline pH, both in control and heat stressed chloroplasts. This result suggests that the alkaline pH accelerates electron donation from secondary electron donor of photosystem II to Q_A both in control and heat stressed samples. Thus the increase in fluorescence level probed by weak modulated light due to preheating is not solely linked to increase in true F₀ level, but largely associated with the shift in the redox state of Q_A, the primary stable electron acceptor of photosystem II.Abbreviations ADRY Acceleration of Deactivation of Reaction of Enzyme Y - CCCP Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone - Chl Chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FeCN potassium ferricyanide - HEPES 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid - LHCP Light harvesting chlorophyll protein - MES (4-morpholine ethane sulfonic acid) - PS photosystem - Q_A and Q_B first and second consecutive electron acceptors of photosystem II - TES (2-[tris(hydroxymethyl)-methylamino]-1-ethanesulfonic acid) sulfonic acid - TRICINE N-[tris(hydroxymethyl)methyl] glycine     

Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition

Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20° C and the fluorescence emission spectra at-196° C indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F ₀ Level of chlorophyll fluorescence when photosystem 2 traps are open - F _m Level of chlorphyll fluorescence when photosystem 2 traps are closed - P Maximum level of fluorescence reached in the absence of DCMU - PSI (II) photosystem I(II)     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

Molecular mechanism of high-temperature-induced inhibition of acceptor side of Photosystem II

High-temperature-induced inhibition of the acceptor side of Photosystem II (PS II) was studied in tobacco thylakoids using oxygen evolution, chlorophyll a (Chl a) fluorescence and redox potential measurements. When thylakoids were heated at 2 °C/min from 25 to 50 °C, the oxygen evolving complex became inhibited between 32 and 45 °C, whereas the acceptor side of PS II tolerated higher temperatures. Variable Chl a fluorescence decreased more slowly than oxygen evolution, suggesting that transitions between some S-states occurred even after heat-induced inhibition of the oxygen evolving activity. 77 K emission spectroscopy reveals that heating does not cause detachment of the light-harvesting complex II from PS II, and thus the heat-induced increase in the initial F₀ fluorescence is due to loss of exciton trapping in the heated PS II centers. Redox titrations showed a heat-induced increase in the midpoint potential of the Q_A/Q_A ^-) couple from the control value of –80 mV to +40 mV at 50 °C, indicating a loss of the reducing power of Q_A ^-). When its driving force thus decreased, electron transfer from Q_A ^-) to Q_B in the PS II centers that still could reduce Q_A became gradually inhibited, as shown by measurements of the decay of Chl a fluorescence yield after a single turnover flash. Interestingly, the heat-induced loss of variable fluorescence and inhibition of electron transfer from Q_A ^-) to Q_B could be partially prevented by the presence of 5 mM bicarbonate during heating, suggesting that high temperatures cause release of the bicarbonate bound to PS II. We speculate that both the upshift in the redox potential of the Q_A/Q_A ^-) couple and the release of bicarbonate may be caused by a heat-induced structural change in the transmembrane D1 or D2 proteins. This structural change may, in turn, be caused by the inhibition of the oxygen evolving complex during heating.     

High photosynthetic efficiency of a rice (Oryza sativa L.) xantha mutant

Comparative analysis revealed that a xantha rice mutant (cv. Huangyu B) had higher ratios of chlorophyll (Chl) a/b and carotenoids/Chl, and higher photosynthetic efficiency than its wild type parent (cv. II32 B). Unexpectedly, the mutant had higher net photosynthetic rate (P _N) than II32 B. This might have resulted from its lower non-photochemical quenching (q_N) but higher maximal photochemical efficiency (F_V/F_M), higher excitation energy capture efficiency of photosystem 2 (PS2) reaction centres (F_V′/F_M′), higher photochemical quenching (q_P), higher effective PS2 quantum yield (Φ_PS2), and higher non-cyclic electron transport rate (ETR). This is the first report of a chlorophyll mutant that has higher photosynthetic efficiency and main Chl fluorescence parameters than its wild type. This mutant could become a unique material both for the basic research on photosynthesis and for the development of high yielding rice cultivars.     

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<Subscript>Fd</Subscript> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F_v, F_m, F₀, F_m′, F_v′, NF, and ΔF, the Chl fluorescence ratios F_v/F_m, F_v/F₀, ΔF/F_m′, as well as the photochemical (q_P) and non-photochemical quenching coefficients (q_N, q_CN, and NPQ). q_N consists of three components (q_N = q_E + q_T + q_I), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q_N, q_CN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q_N was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q_CN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R_Fd (F_d/F_s), which, when measured at saturation irradiance is directly correlated to the net CO₂ assimilation rate (_{P N}) of leaves. This R_Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the R_Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.