<Emphasis Type="Italic">PsbS</Emphasis> genotype in relation to coordinated function of PS II and PS I in <Emphasis Type="Italic">Arabidopsis</Emphasis> leaves

Application of multiple probes to systems that carry specific mutations provides a powerful means for studying how known regulators of light utilization interact in vivo. Two lines of Arabidopsis thaliana were studied, each carrying a unique lesion in the nuclear psbS gene encoding a 22-kDa pigment-binding protein (PS II-S) essential for full expression of photoprotective, rapid-phase, nonphotochemical quenching of chlorophyll fluorescence (NPQ). The PS II-S protein is absent in line npq4-1 due to deletion of psbS. Line npq4-9 expresses normal levels of PS II-S but carries a single amino acid substitution that lowers NPQ capacity by about 50%. A prior report [Peterson RB and Havir EA (2001) Planta 214: 142–152] described an altered pattern of redox states of the acceptor side of Photosystem II (PS II) and donor side of Photosystem I (PS I) for npq4-9 suggesting that interphotosystem electron transport may be restricted by a higher transthylakoid ΔpH in this line. In vivo steady state fluorescence and absorbance measurements (820 nm) confirmed these earlier observations for line npq4-9 but not for npq4-1. Thus, the prior results cannot be correlated simply to a loss of NPQ capacity. Likewise, the kinetics of the 820-nm absorbance change did not indicate a substantial effect of psbS genotype on electron flow from plastoquinol to PS I. A simple model is proposed to relate linear electron transport rate (measured gasometrically) to a parameter (based on fluorescence) that provides a relative measure of the density of excitation available for photochemistry in PS II. Surprisingly, analyses using this model suggested that the in vivo midpoint potential of the primary quinone acceptor in PS II (Q_A) is lowered in both psbS mutant lines. This heretofore-unsuspected role for PS II-S is discussed with regard to: (1) numerous prior reports indicating plasticity of the redox potential of Q_A and (2) the basis for the contrasting regulation of quantum yields of PS I and II in npq4-1 and npq4-9.     

Molecular and global time-resolved analysis of a psbS gene dosage effect on pH- and xanthophyll cycle-dependent nonphotochemical quenching in photosystem II

Photosynthetic light harvesting in plants is regulated by a pH- and xanthophyll-dependent nonphotochemical quenching process (qE) that dissipates excess absorbed light energy and requires the psbS gene product. An Arabidopsis thaliana mutant, npq4-1, lacks qE because of a deletion of the psbS gene, yet it exhibits a semidominant phenotype. Here it is shown that the semidominance is due to a psbS gene dosage effect. Diploid Arabidopsis plants containing two psbS gene copies (wild-type), one psbS gene (npq4-1/NPQ4 heterozygote), and no psbS gene (npq4-1/npq4-1 homozygote) were compared. Heterozygous plants had 56% of the wild-type psbS mRNA level, 58% of the wild-type PsbS protein level, and 60% of the wild-type level of qE. Global analysis of the chlorophyll a fluorescence lifetime distributions revealed three components in wild-type and heterozygous plants, but only a single long lifetime component in npq4-1. The short lifetime distribution associated with qE was inhibited by more than 40% in heterozygous plants compared with the wild type. Thus, the extent of qE measured as either the fractional intensities of the PSII chlorophyll a fluorescence lifetime distributions or steady state intensities was stoichiometrically related to the amount of PsbS protein.     

A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events

The initial (F₀), maximal (F_M) and steady-state (F_S) levels of chlorophyll fluorescence emitted by intact pea leaves exposed to various light intensities and environmental conditions, were measured with a modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the fluorescence signals contain only three terms, X=J₂p_2F/(1–G), Y=T/(1–G) and V, where V is the relative variable fluorescence, J₂ is the light absorption flux in PS II, p_2F is the probability of fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II units and for energy cycling between the reaction center and the chlorophyll pool: F₀=X, F_M=X/(1–Y) and F_S=X(1+(YV/(1–Y))). It is demonstrated that the amplitudes of the previously defined coefficients of chlorophyll fluorescence quenching, q_P and q_N, reflect, not just photochemical (q_P) or nonphotochemical (q_N) events as implied in the definitions, but both photochemical and nonphotochemical processes of PS II deactivation. The coefficient q_P is a measure of the ratio between the actual macroscopic quantum yield of photochemistry in PS II (41-1) in a given light state and its maximal value measured when all PS II traps are open (41-2) in that state, with 41-3 and 41-4. When the partial connection between PS II units is taken into consideration, 1-q_P is nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other hand, 1-q_N equals the (normalized) ratio of the rate constant of photochemistry (k_2b) to the combined rate constant (k_N) of all the nonphotochemical deactivation processes excluding the rate constant k₂₂ of energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the individual rate constants, k_N-k₂₂ and k_2b, is provided by the fluorescence ratios 1/F_M and (1/F₀)–(1/F_M), respectively. Although, in theory, 41-5 is determined by the value of both k_2b and k_N-k₂₂, experimental results presented in this paper show that, under various environmental conditions, 41-6 is modulated largely through changes in k _N, confirming the idea that PS II quantum efficiency is dynamically regulated in vivo by nonphotochemical energy dissipation.Abbreviations Chl chlorophyll - F₀, F_M and F_S initial, maximal and steady-state levels of modulated Chl fluorescence emitted by light-adapted leaves - PS I and II photosystem I and II - q_P and q_N (previously defined) photochemical and nonphotochemical components of Chl fluorescence quenching     

Photoinactivation of photosystem II by cumulative exposure to short light pulses during the induction period of photosynthesis

Photoinactivation of Photosystem (PS) II in vivo was investigated by cumulative exposure of pea, rice and spinach leaves to light pulses of variable duration from 2 to 100 s, separated by dark intervals of 30 min. During each light pulse, photosynthetic induction occurred to an extent depending on the time of illumination, but steady-state photosynthesis had not been achieved. During photosynthetic induction, it is clearly demonstrated that reciprocity of irradiance and duration of illumination did not hold: hence the same cumulative photon exposure (mol m^–2) does not necessarily give the same extent of photoinactivation of PS II. This contrasts with the situation of steady-state photosynthesis where the photoinactivation of PS II exhibited reciprocity of irradiance and duration of illumination (Park et al. (1995) Planta 196: 401–411). We suggest that, for reciprocity to hold between irradiance and duration of illumination, there must be a balance between photochemical (qP) and non-photochemical (NPQ) quenching at all irradiances. The index of susceptibility to light stress, which represents an intrinsic ability of PS II to balance photochemical and non-photochemical quenching, is defined by the quotient (1-qP)/NPQ. Although constant in steady-state photosynthesis under a wide range of irradiance (Park et al. (1995). Plant Cell Physiol 36: 1163–1169), this index of susceptibility for spinach leaves declined extremely rapidly during photosynthetic induction at a given irradiance, and, at a given cumulative photon exposure, was dependent on irradiance. During photosynthetic induction, only limited photoprotective strategies are developed: while the transthylakoid pH gradient conferred some degree of photoprotection, neither D1 protein turnover nor the xanthophyll cycle was operative. Thus, PS II is more easily photoinactivated during photosynthetic induction, a phenomenon that may have relevance for understorey leaves experiencing infrequent, short sunflecks.Abbreviations D1 protein psbA gene product - DTT dithiothreitol - F_v, F_m, F_o variable, maximum, and initial (corresponding to open traps) chlorophyll fluorescence yield, respectively - NPQ non-photochemical quenching - PS Photosystem - Q_A primary quinone acceptor of PS II - qP photochemical quenching coefficient     

Electron transport to oxygen mitigates against the photoinactivation of Photosystem II in vivo

The role of electron transport to O₂ in mitigating against photoinactivation of Photosystem (PS) II was investigated in leaves of pea (Pisum sativum L.) grown in moderate light (250 mol m^–2 s^–1). During short-term illumination, the electron flux at PS II and non-radiative dissipation of absorbed quanta, calculated from chlorophyll fluorescence quenching, increased with increasing O₂ concentration at each light regime tested. The photoinactivation of PS II in pea leaves was monitored by the oxygen yield per repetitive flash as a function of photon exposure (mol photons m^–2). The number of functional PS II complexes decreased nonlinearly with increasing photon exposure, with greater photoinactivation of PS II at a lower O₂ concentration. The results suggest that electron transport to O₂, via the twin processes of oxygenase photorespiration and the Mehler reaction, mitigates against the photoinactivation of PS II in vivo, through both utilization of photons in electron transport and increased nonradiative dissipation of excitation. Photoprotection via electron transport to O₂ in vivo is a useful addition to the large extent of photoprotection mediated by carbon-assimilatory electron transport in 1.1% CO₂ alone.Abbreviations Fm, Fo, Fv- maximal, initial (corresponding to open PS II traps) and variable chlorophyll fluorescence yield, respectively - NPQ- non-photochemical quenching - PS- photosystem - Q_A- primary quinone acceptor - qP- photochemical quenching coefficient     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Short-term responses of Photosystem I to heat stress

When 23°C-grown potato leaves (Solanum tuberosum L.) were exposed for 15 min to elevated temperatures in weak light, a dramatic and preferential inactivation of Photosystem (PS) II was observed at temperatures higher than about 38°C. In vivo photoacoustic measurements indicated that, concomitantly with the loss of PS II activity, heat stress induced a marked gas-uptake activity both in far-red light (>715 nm) exciting only PS I and in broadband light (350–600 nm) exciting PS I and PS II. In view of its suppression by nitrogen gas and oxygen and its stimulation by high carbon-dioxide concentrations, the bulk of the photoacoustically measured gas uptake by heat-stressed leaves was ascribed to rapid carbon-dioxide solubilization in response to light-modulated stroma alkalization coupled to PS I-driven electron transport. Heat-induced gas uptake was observed to be insensitive to the PS II inhibitor diuron, sensitive to the plastocyanin inhibitor HgCl₂ and saturated at a rather high photon flux density of around 1200 E m^–2 s^–1. Upon transition from far-red light to darkness, the oxidized reaction center P700⁺ of PS I was re-reduced very slowly in control leaves (with a half time t_1/2 higher than 500 ms), as measured by leaf absorbance changes at around 820 nm. Heat stress caused a spectacular acceleration of the postillumination P700⁺ reduction, with t_1/2 falling to a value lower than 50 ms (after leaf exposure to 48°C). The decreased t_1/2 was sensitive to HgCl₂ and insensitive to diuron, methyl viologen (an electron acceptor of PS I competing with the endogenous acceptor ferredoxin) and anaerobiosis. This acceleration of the P700⁺ reduction was very rapidly induced by heat treatment (within less than 5 min) and persisted even after prolonged irradiation of the leaves with far-red light. After heat stress, the plastoquinone pool exhibited reduction in darkness as indicated by the increase in the apparent Fo level of chlorophyll fluorescence which could be quenched by far-red light. Application (for 1 min) of far-red light to heat-pretreated leaves also induced a reversible quenching of the maximal fluorescence level Fm, suggesting formation of a pH gradient in far-red light. Taken together, the presented data indicate that PS I responded to the heat-induced loss of PS II photochemical activity by catalyzing an electron flow from stromal reductants. Heat-stress-induced PS I electron transport independent of PS II seems to constitute a protective mechanism since block of this electron pathway in anaerobiosis was observed to result in a dramatic photoinactivation of PS I.Abbreviations PFD photon flux density - PS Photosystem - Apt and Aox amplitude of the photothermal and photobaric components of the photoacoustic signal, respectively - P700 reaction center pigment of PS I - Fo and Fm initial and maximal levels of chlorophyll fluorescence, respectively - Fv=Fm Fo-variable chlorophyll fluorescence - Q_A primary (stable) electron acceptor of PS II - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Cyt cytochrome     

Coregulation of electron transport through PS I by Cyt b 6 f,excitation capture by P700 and acceptor side reduction. Time kinetics and electron transport requirement

Regulation of electron transport rate through Photosystem I (PS I) was investigated in intact sunflower leaves. The rate constant of electron donation via the cytochrome b ₆ f complex (k_q, s^–1) was obtained from the postillumination P700⁺ reduction rate, measured as the exponential decay of the light-dark difference (D830) of the 830 nm transmission signal. D830 corresponding to maximum oxidisable P700 (D830_m) was obtained by applying white light flashes of different intensity and extrapolating the plot of the quantum yield Y vs. D830 to the axis of abscissae (Y->0). Maximum quantum yield of PS I at completely reduced P700 (Y_m) was obtained by extrapolating the same plot to the axis of ordinates (D830->0). Regulation of k_q, D830_m and Y_m under rate-limiting CO₂ and O₂ concentrations applied after air (21% O₂, 310 ppm CO₂) was investigated. The amplitude of the downregulation of k_q (photosynthetic control) was maximal when electron transport rate (ETR) was limited to about 3 nmol cm^–2 s^–1 and decreased when ETR was higher or lower. Downregulation did not occur in the absence of CO₂ and O₂. These gases acted only as substrates of ribulosebisphosphate carboxylase-oxygenase, no high-affinity reaction of O₂ leading to enhanced photosynthetic control (e.g. Mehler reaction) was detected. After the transition, D830_m at first decreased and then increased again, showing that the reduction of the PS I acceptor side disappeared as a result of the downregulation of k_q. The variation of Y_m had two reasons, PS I acceptor side reduction and variable excitation capture efficiency by P700. It is concluded that electron transport through PS I is coregulated by the rate of plastoquinol oxidation at Cyt b ₆ f, excitation capture efficiency by P700, and by acceptor side reduction.Abbreviations Cyt b ₆ f cytochrome b ₆ f complex - D830 difference of the 830 nm signal from the dark level - ETR electron transport rate - PAD photon absorption density nmol cm^–2 s^–1 - PFD incident photon flux density, nmol cm^–2 s^–1 - PS I Photosystem I - PS II Photosystem II - PQH₂ plastoquinol - P700 Photosystem I donor pigment - Y quantum yield of PS I electron transport, rel. un.     

LHC II protein phosphorylation in leaves of Arabidopsis thaliana mutants deficient in non-photochemical quenching

Phosphorylation of the light-harvesting chlorophyll a/b complex II (LHC II) proteins is induced in light via activation of the LHC II kinase by reduction of cytochrome b₆f complex in thylakoid membranes. We have recently shown that, besides this activation, the LHC II kinase can be regulated in vitro by a thioredoxin-like component, and H₂O₂ that inserts an inhibitory loop in the regulation of LHC II protein phosphorylation in the chloroplast. In order to disclose the complex network for LHC II protein phosphorylation in vivo, we studied phosphorylation of LHC II proteins in the leaves of npq1-2 and npq4-1 mutants of Arabidopis thaliana. In comparison to wild-type, these mutants showed reduced non-photochemical quenching and increased excitation pressure of Photosystem II (PS II) under physiological light intensities. Peculiar regulation of LHC II protein phosphorylation was observed in mutant leaves under illumination. The npq4-1 mutant was able to maintain a high amount of phosphorylated LHC II proteins in thylakoid membranes at light intensities that induced inhibition of phosphorylation in wild-type leaves. Light intensity-dependent changes in the level of LHC II protein phosphorylation were smaller in the npq1-2 mutant compared to the wild-type. No significant differences in leaf thickness, dry weight, chlorophyll content, or the amount of LHC II proteins were observed between the two mutant and wild-type lines. We propose that the reduced capacity of the mutant lines to dissipate excess excitation energy induces changes in the production of reactive oxygen species in chloroplasts, which consequently affects the regulation of LHC II protein phosphorylation.     

Very high light resistant mutants of Chlamydomonas reinhardtii: Responses of Photosystem II,nonphotochemical quenching and xanthophyll pigments to light and CO2

We have isolated very high light resistant nuclear mutants (VHL ^R) in Chlamydomonas reinhardtii, that grow in 1500–2000 mol photons m^–2 s^–1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL ^R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L ^*) from which another class of VHL ^R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL ^R mutants was independent of CO₂ availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mol photons m^–2 s^–1) was strongly depressed when all genotypes were grown in 5% CO₂. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO₂, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid pH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO₂ at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L ^* and VHL ^R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO₂, possibly because they were unable to maintain sufficiently high pH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL ^R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL ^R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.This revised version was published online in October 2005 with corrections to the Cover Date.     

Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes,fluorescence and photosynthesis in leaves of Hedera canariensis

The role of the xanthophyll cycle in regulating the energy flow to the PS II reaction centers and therefore in photoprotection was studied by measurements of light-induced absorbance changes, Chl fluorescence, and photosynthetic O₂ evolution in sun and shade leaves of Hedera canariensis. The light-induced absorbance change at 510 nm (A₅₁₀) was used for continuous monitoring of zeaxanthin formation by de-epoxidation of violaxanthin. Non-radiative energy dissipation (NRD) was estimated from non-photochemical fluorescence quenching (NPQ).High capacity for zeaxanthin formation in sun leaves was accompanied by large NRD in the pigment bed at high PFDs as indicated by a very strong NPQ both when all PS II centers are closed (F'_m) and when all centers are open (F'_o). Such F_o quenching, although present, was less pronounced in shade leaves which have a much smaller xanthophyll cycle pool.Dithiothreitol (DTT) provided through the cut petiole completely blocked zeaxanthin formation. DTT had no detectable effect on photosynthetic O₂ evolution or the photochemical yield of PS II in the short term but fully inhibited the quenching of F_o and 75% of the quenching of F_m, indicating that NRD in the antenna was largely blocked. This inhibition of quenching was accompanied by an increased closure of the PS II reaction centers.In the presence of DTT a photoinhibitory treatment at a PFD of 200 mol m^-2 s^-1, followed by a 45 min recovery period at a low PFD, caused a 35% decrease in the photon yield of O₂ evolution, compared to a decrease of less than 5% in the absence of DTT. The F_v/F_m ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT F_o rose by 15–20% whereas no change was detected in control leaves.The results support the conclusion that the xanthophyll cycle has a central role in regulating the energy flow to the PS II reaction centers and also provide direct evidence that zeaxanthin protects against photoinhibitory injury to the photosynthetic system.Abbreviations F, F_m, F_o, F_v Fluorescence yield at actual degree of PS II center closure, when all centers are closed, when all centers are open, variable fluorescence - NPQ non-photochemical fluorescence quenching - NRD non-radiative energy dissipation - PFD photon flux density - Q_A primary acceptor PS II     

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     

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (–196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 mol O₂ per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable fluorescence - LHC light-harvesting complex - PpBQ phenyl-p-benzoquinone - PQ plastoquinone pool - P700 reaction center of PS I - PS I, PS II Photosystem I, II - Q_A first bound plastoquinone accepter - RC reaction centre     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Determination of the quantum efficiency of photosystem II and of non-photochemical quenching of chlorophyll fluorescence in the field

A newly developed portable chlorophyll fluorometer in combination with a special leaf clip holder was used for assessing photosynthetic activity of attached sun leaves of Fagus sylvatica and Cucurbita pepo under field conditions. During diurnal time courses, fluorescence yield, photosynthetic photon flux density (PPFD) incident on the leaf plane, and leaf temperature were measured and quantum efficiency of photosystem II (PS II), apparent relative electron transport rates, and non-photochemical fluorescence quenching (NPQ) calculated. In both species, quantum efficiency followed closely the incident PPFD and no hysteresis could be observed during the day. Apparent electron transport rate showed light saturation above a PPFD of 700 mol m^–2 s^–1 in F. sylvatica, while in C. pepo no saturation was visible up to 1400 mol m^–2 s^–1. NPQ was closely correlated to excessive PPFD calculated from the PS II quantum yield. Maximal NPQ observed was 3.3 Although the beech leaf was exposed for a considerable time to PPFD values of 1400–1500 mol m^–2 s^–1 and leaf temperatures between 30 and 35°C, no obvious signs for sustained photodamage could be observed. The data demonstrate the potential of chlorophyll fluorescence measurements to analyse photosynthetic performance under field conditions with minimal disturbance of the plant. Potential error sources due to the geometry of the leaf clip holder used are discussed.Dedicated to Prof. Dr. F.-C. Czygan on the occasion of his 60th birthday     

Reduced levels of cytochrome b 6/f in transgenic tobacco increases the excitation pressure on Photosystem II without increasing sensitivity to photoinhibition in vivo

We have examined tobacco transformed with an antisense construct against the Rieske-FeS subunit of the cytochromeb ₆ f complex, containing only 15 to 20% of the wild-type level of cytochrome f. The anti-Rieske-FeS leaves had a comparable chlorophyll and Photosystem II reaction center stoichiometry and a comparable carotenoid profile to the wild-type, with differences of less than 10% on a leaf area basis. When exposed to high irradiance, the anti-Rieske-FeS leaves showed a greatly increased closure of Photosystem II and a much reduced capacity to develop non-photochemical quenching compared with wild-type. However, contrary to our expectations, the anti-Rieske-FeS leaves were not more susceptible to photoinhibition than were wild-type leaves. Further, when we regulated the irradiance so that the excitation pressure on photosystem II was equivalent in both the anti-Rieske-FeS and wild-type leaves, the anti-Rieske-FeS leaves experienced much less photoinhibition than wild-type. The evidence from the anti-Rieske-FeS tobacco suggests that rapid photoinactivation of Photosystem II in vivo only occurs when closure of Photosystem II coincides with lumen acidification. These results suggest that the model of photoinhibition in vivo occurring principally because of limitations to electron withdrawal from photosystem II does not explain photoinhibition in these transgenic tobacco leaves, and we need to re-evaluate the twinned concepts of photoinhibition and photoprotection.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlophenyl)-1,-dimethylurea - Fo and Fo minimal fluorescence when all PS II reaction centers are open in dark- and light-acclimated leaves, respectively - Fm and Fm maximal fluorescence when all PS II reaction centers are closed in dark- and light-acclimated leaves, respectively - Fv variable fluorescence (Fm-Fo) in dark acclimated leaves - Fv variable fluorescence (Fm-Fo) in lightacclimated leaves - NPQ non-photochemical quenching of fluorescence - PS I and PS II Photosystem I and II - P680 primary electron donor of the reaction center of PS II - PFD photosynthetic flux density - Q_A primary acceptor quinone of PS II - q_p photochemical quenching of fluorescence - V+A+Z violaxanthin+antheraxanthin+zeaxanthin     

Photoinhibition and Recovery of photosystem 2 in Grapevine (Vitis vinifera L.) Leaves Grown Under Field Conditions

Photoinhibition of photosynthesis was investigated in grapevine (Vitis vinifera L.) exposed to 2 or 4h of high irradiance (HI) (1 700–1 800 mol m^–2 s^–1) leaves under field conditions at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and photosynthetic electron transport measurements. When the photochemical efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined, F₀ increased in both 2 (HI2) and 4 h (HI4) HI leaves sampled at midday. When various photosynthetic activities were followed on isolated thylakoids, HI4 leaves showed significantly higher inhibition of whole chain and PS2 activity than the HI2 leaves sampled at midday. Later, the leaves reached maximum PS2 efficiencies similar to those observed early in the morning during sampling at evening. The artificial exogenous electron donor Mn²⁺ failed to restore PS2 activity in both variants of leaves, while DPC and NH²OH significantly restored PS2 activity in HI4 midday leaf samples. Quantification of the PS2 reaction centre protein D1 and 33 kDa protein of water splitting complex following midday exposure of leaves showed pronounced differences between HI2 and HI4 leaves. The marked loss of PS2 activity noticed in midday samples was mainly due to the marked loss of D1 protein in HI2, while in HI4 it was mainly 33-kDa protein.     

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S₂₍₃₎Q_B ^– (B band), S₂Q_A ^– (Q band), Tyr-D⁺Q_A ^– (C band) and Tyr-Z⁺(His⁺)Q_A ^– (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S₂₍₃₎Q_B ^– recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DT desiccation tolerant - PS II Photosystem II - TL thermoluminescence - P₆₈₀ reaction center Chl of PS II - Q_A and Q_B puinone electron acceptors of PS II - S₀,...,S₄ the redox states of the water-oxidizing complex - Tyr-Z and Tyr-D redox-active tyrosine electron donors of PS II     

Light acclimation maintains the redox state of the PS II electron acceptor Q2 within a narrow range over a broad range of light intensities

Chrysanthemum inducum-hybrid `Coral Charm', Hibiscus rosa-sinensis L. `Cairo Red' and Spathiphyllum wallisii Regel `Petit' were grown in natural light in a greenhouse at three levels of irradiance using permanent shade screens. Light acclimation of photosynthesis was characterized using modulated chlorophyll a fluorescence of intact leaves. A close correlation was found between the degree of reduction of the primary electron acceptor Q_A of Photosystem II (PS II) approximated as the fluorescence parameter 1−q_P, and light acclimation. The action range of 1−q_P was 0–0.4 from darkness to full irradiance around noon, within the respective light treatments in the greenhouse, indicating that most PS II reaction centres were kept open. In general, the index for electron transport (ETR) measured by chlorophyll fluorescence was higher for high-light (HL) than intermediate-(IL) and low-light (LL) grown plants. However, HL Chrysanthemum showed 40% higher ETR than HL Hibiscus at light saturation, despite identical redox states of Q_A. The light acclimation of the non-radiative dissipation of excess energy in the antenna, NPQ, varied considerably between the species. However, when normalized against q_P, a strong negative correlation was found between thermal dissipation and ETR measured by chlorophyll fluorescence. To be able to accommodate a high flux of electrons through PS II, the plants with the highest light-saturated ETR had the lowest NPQ/q_P. The possibility of using chlorophyll fluorescence for quantification of the energy balance between energy input and utilization in PS II in intact leaves is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoprotection in a zeaxanthin- and lutein-deficient double mutant of Arabidopsis

When light absorption by a plant exceeds its capacity for light utilization, photosynthetic light harvesting is rapidly downregulated by photoprotective thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). To address the involvement of specific xanthophyll pigments in NPQ, we have analyzed mutants affecting xanthophyll metabolism in Arabidopsis thaliana. An npq1 lut2 double mutant was constructed, which lacks both zeaxanthin and lutein due to defects in the violaxanthin de-epoxidase and lycopene -cyclase genes. The npq1 lut2 strain had normal Photosystem II efficiency and nearly wild-type concentrations of functional Photosystem II reaction centers, but the rapidly reversible component of NPQ was completely inhibited. Despite the defects in xanthophyll composition and NPQ, the npq1 lut2 mutant exhibited a remarkable ability to tolerate high light.This revised version was published online in October 2005 with corrections to the Cover Date.