The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves

It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that non-photochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.     

Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves

A new type of modulation fluorometer was used in the study of energy-dependent chlorophyll fluorescence quenching (q_E) in intact leaves. Under conditions of strong energization of the thylakoid membrane (high light intensity, absence of CO₂) not only variable fluorescence, F_V, but also dark-level fluorescence, F_O, was quenched, leading to definition of a quenching coefficient, q_O. Information on q_O was shown to be essential for correct determination of photochemical (q_Q) and energy dependent quenching (q_E) by the saturation pulse method. The relationship between q_E and q_O was analysed over a range of light intensities at steady state conditions. q_E was found to consist of two components, the second of which is linearly correlated with q_O. q_O and the second component of q_E are interpreted to reflect the state 1 — state 2 shift caused by LHC II phosphorylation.     

Cryptic chlorophyll breakdown in non-senescent green Arabidopsis thaliana leaves

Photosynthesis Research - Chlorophyll (Chl) breakdown is a diagnostic visual process of leaf senescence, which furnishes phyllobilins (PBs) by the PAO/phyllobilin pathway. As Chl breakdown disables...     

Photoinhibition, 77K chlorophyll fluorescence quenching and phosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in soybean leaves

When the capacity of leaves for orderly dissipation of excitation energy in photosynthesis is exceeded, one mechanism by which the excess energy appears to be dissipated is through a nonradiative decay process. This process is observed as a reversible quenching of chlorophyll fluorescence emission (77K) from both photosystem II and photosystem I which persists in darkness (Demmig and Björkman 1987, Planta 171, 171–184). Fluorescence quenching was induced in soybean (Glycine max (L.) Merr.) leaves by two methods: 1) changing the composition of the gas surrounding the leaf from normal air to 2% O₂, 0% CO₂ at a low, constant photon flux density (PFD=photon fluence rate), and 2) increasing the PFD in the presence of normal air. In either case the quenching was fully reversible after return to the original condition (low PFD, normal air). The half-time of the relaxation of the quenching was in the order of 30 min. Both treatments resulted in reversible dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II (LHC-II). Treatment under photoinhibitory conditions (high PFD plus chloramphenicol) also caused dephosphorylation of LHC-II. Therefore, phosphorylation of LHC-II cannot account for the observed fluorescence quenching. In addition, our results indicate that in vivo a factor other than the redox state of the plastoquinone pool controls LHC-II phosphorylation. This factor may be pH, the pH gradient across the thylakoid membranes.Abbreviations and symbols CAP chloramphenicol - F_o, F_M, F_v instantaneous, maximumr variable fluorescence emission - LHC-II light-haryesting chlorophyll-protein complex of PSII - kDa kilodalton - pH pH gradient across the thylakoid membrane - PFD photon flux density (photon fluence rate) - PQ plastoquinone - PSI, PSII photosystem I, II - Q acceptor of PSII C.I.W.-D.P.B. Publication No. 926     

Resolution of components of non-photochemical chlorophyll fluorescence quenching in barley leaves

Non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves has been analysed by monitoring its relaxation in the dark, by applying saturating pulses of light. At least three kinetically distinct phases to qN recovery are observed, which have previously been identified (Quick and Stitt 1989) as being due to high-energy state quenching (fast), excitation energy redistribution due to a state transition (medium) and photoinhibition (slow). However, measurements of chlorophyll fluorescence at 77 K from leaf extracts show that state transitions only occur in low light conditions, whereas the medium component of qN is very large in high light. The source of that part of the medium component not accounted for by a state transition is discussed.Abbreviations ATP adenosine 5-triphosphate - DCMU 3[3,4-dichlorophenyl]-1,1 dimethylurea - pH trans-thylakoid pH gradient - F_o, F_m room-temperature chlorophyll fluorescence yield with all reaction centres open, closed - F_v variable fluorescence = F_m–F_o - LHC II Light harvesting complex II - PS I, PS II Photosystem I, II - P700, P680 primary donor in photosystem I, II - qP photochemical quenching of variable fluorescence - qN non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i non-photochemical quenching due to high energy state, state transition, photoinhibition - qN_f, qN_m, qN_s components of qN relaxing fast, medium, slow - q_r quenching of r relative to the dark state - tricine N-tris[hydroxymethyl]methylglycine - r ratio of fluorescence maximum from photosystem II to that from photosystem I at 77 K     

Evidence for variable chlorophyll fluorescence of photosystem I in vivo

Photosynthesis Research - Room temperature fluorescence in vivo and its light-induced changes are dominated by chlorophyll a fluorescence excited in photosystem II, F(II), peaking around...     

Identification of a single-copy gene encoding a Type I chlorophyll a/b-binding polypeptide of photosystem I in Arabidopsis thaliana

We have isolated and sequenced cDNA and genomic clones from Arabidopsis thaliana which specify a 241 residue protein with 84% sequence identity to a photosystem I Type I chlorophyll a/b -binding (CAB) protein from tomato. The open reading frame is interrupted by three introns which are found at equivalent positions as the corresponding introns in the tomato gene. Comparison to the amino acid sequence of other CAB proteins confirms that all CAB proteins share two regions of very high similarity. However, near the N-terminus and between the conserved regions this light-harvesting complex I (LHCI) protein, as other LHCI proteins from other plant species, has sequence motifs which appear to be PSI-specific. Restriction analysis of genomic DNA shows that the Arabidopsis protein is encoded by a single-copy gene.     

Deriving fluorometer-specific values of relative PSI fluorescence intensity from quenching of F 0 fluorescence in leaves of Arabidopsis thaliana and Zea mays

The effect of stepwise increments of red light intensities on pulse-amplitude modulated (PAM) chlorophyll (Chl) fluorescence from leaves of A. thaliana and Z. mays was investigated. Minimum and maximum fluorescence were measured before illumination (F ₀ and F _M, respectively) and at the end of each light step ( $ F^{\prime}_{0} $ and $ F^{\prime}_{\text{M}} $ , respectively). Calculated $ F^{\prime}_{0} $ values derived from F ₀, F _M and $ F^{\prime}_{\text{M}} $ fluorescence according to Oxborough and Baker (1997) were lower than the corresponding measured $ F^{\prime}_{0} $ values. Based on the concept that calculated $ F^{\prime}_{0} $ values are under-estimated because the underlying theory ignores PSI fluorescence, a method was devised to gain relative PSI fluorescence intensities from differences between calculated and measured $ F^{\prime}_{0} $ . This method yields fluorometer-specific PSI data as its input data (F ₀, F _M, $ F^{\prime}_{0} $ and $ F^{\prime}_{\text{M}} $ ) depend solely on the spectral properties of the fluorometer used. Under the present conditions, the PSI contribution to F ₀ fluorescence was 0.24 in A. thaliana and it was independent on the light acclimation status; the corresponding value was 0.50 in Z. mays. Correction for PSI fluorescence affected Z. mays most: the linear relationship between PSI and PSII photochemical yields was clearly shifted toward the one-to-one proportionality line and maximum electron transport was increased by 50 %. Further, correction for PSI fluorescence increased the PSII reaction center-specific parameter, 1/F ₀ ? 1/F _M, up to 50 % in A. thaliana and up to 400 % in Z. mays.     

Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes

Chlorophyll fluorescence quenching can be stimulated in vitro in purified photosystem II antenna complexes. It has been shown to resemble nonphotochemical quenching observed in isolated chloroplasts and leaves in several important respects, providing a model system for study of the mechanism of photoprotective energy dissipation. The effect of temperature on the rate of quenching in trimeric and monomeric antenna complexes revealed the presence of two temperature-dependent processes with different activation energies, one between approximately 15 and 35 degrees C and another between approximately 40 and 60 degrees C. The temperature of the transition between the two phases was higher for trimers than for monomers. Throughout this temperature range, the quenching was almost completely reversible, the protein CD was unchanged, and pigment binding was maintained. The activation energy for the low temperature phase was consistent with local rearrangements of pigments within some of the protein domains, whereas the higher temperature phase seemed to arise from large scale conformational transitions. For both phases, there was a strong linear correlation between the quenching rate and the appearance of an absorption band at 685 nm. In addition, quenching was correlated with a loss of CD at approximately 495 nm from Lutein 1 and at 680 nm from chlorophylls a1 and a2, the terminal emitters. The results obtained indicate that quenching of chlorophyll fluorescence in antenna complexes is brought about by perturbation of the lutein 1/chlorophyll a1/chlorophyll a2 locus, forming a poorly fluorescing chlorophyll associate, either a dimer or an excimer.     

Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence

Regulation of nonradiative dissipation of absorbed light energy in PSII is an indispensable process to avoid photoinhibition in plants. To dissect molecular mechanisms of the regulation, we identified Arabidopsis mutants with reduced quenching of Chl fluorescence using a fluorescence imaging system. By analyses of Chl fluorescence induction pattern in the light and quantum yield of both photosystems, 37 mutants were classified into three groups. The first group was characterized by an extremely high level of minimum Chl fluorescence at the open PSII center possibly due to a defect in PSII. Mutants with significant reduction in the nonphotochemical quenching formation but not in quantum yield of both photosystems were classified into the second group. Mutants in the third group showed reduction in quantum yield of both photosystems possibly due to a defect in the electron transport activity. Mutants in the second and third groups were further characterized by light intensity dependence of Chl fluorescence parameters and steady state redox level of P700.     

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.     

Decreased stability of photosystem I in dgd1 mutant of Arabidopsis thaliana

The dgd1 mutant of Arabidopsis thaliana provides us with a powerful tool for revealing the specific role of digalactosyldiacylglycerol (DGDG) in photosynthesis. Blue-native polyacrylamide gel electrophoresis analysis revealed that photosystem I (PSI) subunits are assembled into a PSI complex, and that a PSI subcomplex lacking stroma side subunits was also present. PSI subunits in the dgd1 mutant were decreased to a similar level compared with that in the wild type (WT) Arabidopsis. Further experiments showed that PSI subunits in the stroma side, PsaD and PsaE, in the dgd1 mutant were more susceptible to removal by chaotropic agents than those in the WT plant, indicating that the stability of PsaD and PsaE is impaired in the dgd1 mutant. These results provide evidence that DGDG is important for the stability of the PSI complex.     

A pulse-amplitude modulated fluorescence-based method for assessing the effects of photosystem II herbicides on freshwater periphyton

A test was developed that measures in vivo chlorophyll afluorescence variables to assess the apparent sensitivity of freshwaterperiphytic algae to photosystem II inhibitors. Natural periphyticcommunities from rivers were collected on artificial substrata, and theeffects of short-term exposures to two PSII herbicides (atrazine andisoproturon) on the fluorescence parameters were measured with apulse-amplitude modulated fluorometer. The EC50 for each herbicide werecalculated from fluorescence yield indices, and these results were comparedto ¹⁴C-based primary production measurements on the samecommunities. The fluorescence-based method appears to give very reliableestimations of EC50 for each pesticide we tested, ranging from 0.46 to5.18 M and 0.07 to 6.77 M for atrazine and isoproturon,respectively. This method could be used in ecotoxicology monitoringprograms, to detect changes in natural periphyton populations sensitivity,following photosystem II herbicide contamination in rivers or lakes.     

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     

Antisense inhibition of the photosystem I antenna protein Lhca4 in Arabidopsis thaliana.

The function of Lhca4, a gene encoding the photosystem 1 type IV chlorophyll a/b-binding protein complex in Arabidopsis, was investigated using antisense technology. Lhca4 protein was reduced in a number of mutant lines and abolished in one. The inhibition of protein was not correlated with the inhibition of mRNA. No depletion of Lhca1 was observed, but the low-temperature fluorescence emission spectrum was drastically altered in the mutants. The emission maximum was blue-shifted by 6 nm, showing that chlorophyll molecules bound to Lhca4 are responsible for most of the long-wavelength fluorescence emission. Some mutants also showed an unexplainable delay in flowering time and an increase in seed weight.     

Simultaneous measurement of stomatal conductance, non-photochemical quenching, and photochemical yield of photosystem II in intact leaves by thermal and chlorophyll fluorescence imaging

A new imaging system capable of simultaneously measuring stomatal conductance and fluorescence parameters, non-photochemical quenching (NPQ) and photochemical yield of photosystem II (Phi(PSII)), in intact leaves under aerobic conditions by both thermal imaging and chlorophyll fluorescence imaging was developed. Changes in distributions of stomatal conductance and fluorescence parameters across Phaseolus vulgaris L. leaves induced by abscisic acid treatment were analyzed. A decrease in stomatal conductance expanded in all directions from the treatment site, then mainly spread along the lateral vein toward the leaf edge, depending on the ABA concentration gradient and the transpiration stream. The relationships between stomatal conductance and fluorescence parameters depended on the actinic light intensity, i.e. NPQ was greater and Phi(PSII) was lower at high light intensity. The fluorescence parameters did not change, regardless of stomatal closure levels at a photosynthetically active photon flux (PPF) of 270 micro mol m(-2) s(-1); however, they drastically changed at PPF values of 350 and 700 micro mol m(-2) s(-1), when the total stomatal conductance decreased to less than 80 and 200 mmol m(-2) s(-1), respectively. This study has, for the first time, quantitatively analyzed relationships between spatiotemporal variations in stomatal conductance and fluorescence parameters in intact leaves under aerobic conditions.     

Photoinhibition of photosystem I at chilling temperature and subsequent recovery in Arabidopsis thaliana

Chilling-induced photoinhibition and subsequent recovery was studied in Arabidopsis thaliana exposed to 4 degrees C and 150 micromol photons m(-2) s(-1). PSII showed progressive damage with a 14% decrease in quantum yield after 8 h exposure. In contrast, the damage to PSI leveled off after 8 h with a decrease in in vitro NADP+ photoreduction activity of around 32%. In vivo P700 measurements demonstrated that antenna efficiency was decreased by the photoinhibitory treatment. Measurements of P700 and immunoblotting demonstrated that the damaged PSI was not degraded during the 8 h light-chilling treatment, but after 12 h recovery at 20 degrees C, no damaged PSI remained in the thylakoids. Thus, degradation of damaged PSI is a step in the recovery and not a direct result of photodamage. Unlike photodamaged PSII, the PSI core complex is not repaired but completely degraded. In contrast, light harvesting complex I proteins have a slow turnover. PSII recovered completely within 8 h after transfer to 20 degrees C whereas PSI activity recovered very slowly, and the amount of PSI on a leaf area basis remained low even after 1 week at 20 degrees C. The results show that damage, protein turnover and recovery are well separated processes in Arabidopsis.     

Role of the two PsaE isoforms on O2 reduction at photosystem I in Arabidopsis thaliana

Leaves of Arabidopsis thaliana plants grown in short days (8 h light) generate more reactive oxygen species in the light than leaves of plants grown in long days (16 h light). The importance of the two PsaE isoforms of photosystem I, PsaE1 and PsaE2, for O₂ reduction was studied in plants grown under these different growth regimes. In short day conditions a mutant affected in the amount of PsaE1 (psae1-1) reduced more efficiently O₂ than a mutant lacking PsaE2 (psae2-1) as shown by spin trapping EPR spectroscopy on leaves and by following the kinetics of P700⁺ reduction in isolated photosystem I. In short day conditions higher O₂ reduction protected photosystem II against photoinhibition in psae1-1. In contrast in long day conditions the presence of PsaE1 was clearly beneficial for photosynthetic electron transport and for the stability of the photosynthetic apparatus under photoinhibitory conditions. We conclude that the two PsaE isoforms have distinct functions and we propose that O₂ reduction at photosystem I is beneficial for the plant under certain environmental conditions.     

Use of simultaneous analysis of gas-exchange and chlorophyll fluorescence quenching for analysing the effects of water stress on photosynthesis in apple leaves

Summary A convenient system for the rapid simultaneous measurement of both chlorophyll fluorescence quenching using a modulated light system, and of CO₂, and water vapour exchange by leaves is described. The system was used in a study of the effects of water deficits on the photosynthesis by apple leaves (Malus x domestica Borkh.). Apple leaves were found to have low values of steady-state variable fluorescence, and the existence of significant fluorescence with open traps (F_o) quenching necessitated the measurement and use of a corrected F_o in the calculation of quenching components. Long-term water stress had a marked effect on both gas-exchange and chlorophyll fluorescence quenching. Non-photochemical quenching (qn) in particular was increased in water-stressed leaves, and it was particularly sensitive to incident radiation in such leaves. In contrast, rapid dehydration only affected gas exchange. Relaxation of qn quenching in the dark was slow, taking approximately 10 min for a 50% recovery, in well-watered and in draughted plants, and whether or not the plants had been exposed to high light.