Excited Chlorophyll Research Articles

Pigment composition and functional state of the thylakoid membranes during preparation of samples for pigment-protein complexes separation by nondenaturing gel electrophoresis

The present study was conducted to examine changes in photosynthetic pigment composition and functional state of the thylakoid membranes during the individual steps of preparation of samples that are intended for a separation of pigmentprotein complexes by nondenaturing polyacrylamide gel electrophoresis. The thylakoid membranes were isolated from barley leaves (Hordeum vulgare L.) grown under low irradiance (50 μmol m−2 s−1). Functional state of the thylakoid membrane preparations was evaluated by determination of the maximal photochemical efficiency of photosystem (PS) II (FV/FM) and by analysis of excitation and emission spectra of chlorophyll a (Chl a) fluorescence at 77 K. All measurements were done at three phases of preparation of the samples: (1) in the suspensions of osmotically-shocked broken chloroplasts, (2) thylakoid membranes in extraction buffer containing Tris, glycine, and glycerol and (3) thylakoid membranes solubilized with a detergent decyl-β-D-maltosid. FV/FM was reduced from 0.815 in the first step to 0.723 in the second step and to values close to zero in solubilized membranes. Pigment composition was not pronouncedly changed during preparation of the thylakoid membrane samples. Isolation of thylakoid membranes affected the efficiency of excitation energy transfer within PSII complexes only slightly. Emission and excitation fluorescence spectra of the solubilized membranes resemble spectra of trimers of PSII light-harvesting complexes (LHCII). Despite a disrupted excitation energy transfer from LHCII to PSII antenna core in solubilized membranes, energy transfer from Chl b and carotenoids to emission forms of Chl a within LHCII trimers remained effective.

Enhanced Photoprotection by Protein-Bound vs Free Xanthophyll Pools: A Comparative Analysis of Chlorophyll b and Xanthophyll Biosynthesis Mutants

When light absorbed by plants exceeds the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly de-epoxidized to zeaxanthin in the so-called xanthophyll cycle. Zeaxanthin plays a key role in the protection of photosynthetic organisms against excess light, by promoting rapidly reversible (qE) and long-term (qI) quenching of excited chlorophylls, and preventing lipid oxidation. The photoprotective role of zeaxanthin, either free or bound to light-harvesting complexes (Lhcs), has been investigated by using mutants lacking Chl b (ch1) and/or specific xanthophyll species (npq, lut2). The ch1 mutation causes (1) the absence of Lhcb proteins; (2) strong reduction of the feedback de-excitation (qE); and (3) accumulation of xanthophylls as free pigments into thylakoids. Ch1 mutants showed extreme sensitivity to photo-oxidative stress in high light, due to higher singlet oxygen (¹O₂) release. The double mutant ch1npq1 was more sensitive to photo-oxidation than ch1, showing that zeaxanthin does protect lipids even when free in the membrane. Nevertheless, lack of zeaxanthin had a much stronger impact on the level of lipid peroxidation in Lhcs-containing plants (WT vs npq1) with respect to Lhc-less plants (ch1 vs ch1npq1), implying that its protective effect is enhanced by interaction with antenna proteins. It is proposed that the antioxidant capacity of zeaxanthin is empowered in the presence of PSII-LHCs-Zea complexes, while its effect on enhancement of qE only provides a minor contribution. Comparison of the sensitivity of WT vs npq1 plants to exogenous ¹O₂ suggests that besides the scavenging of ¹O₂, at least one additional mechanism is involved in chloroplast photoprotection.

The excitation energy transfer in the trimeric fucoxanthin–chlorophyll protein from Cyclotella meneghiniana analyzed by polarized transient absorption spectroscopy

Polarized transient absorption spectroscopy has been applied to study the carotenoid to chlorophyll excitation energy transfer in the trimeric fucoxanthin–chlorophyll protein FCPa of the centric diatom Cyclotella meneghiniana. We examined the transfer pathways after excitation in the main carotenoid band (S 0 → S 2 transition) with two excitation wavelengths that address either red fucoxanthins only or blue fucoxanthins and the xanthophyll cycle pigments. We were able to identify different transition dipole moments for the S 1 and the ICT state, which are assumed to be a single coupled state that transfers excitation energy to chlorophyll a. Furthermore we obtained different transition dipole moments for the first excited state S 1 of fucoxanthin depending on the excitation wavelength.

A revised energy partitioning approach to assess the yields of non-photochemical quenching components

Non-photochemical quenching (NPQ) is a complex and still unclear mechanism essential for higher plants. The intensive research on this subject has highlighted three main components of NPQ: energy-dependent process (qE); state transitions to balance the excitation of PSII and PSI (qT); and photoinhibitory processes (qI). Recently, these components have been resolved as quantum yields according to the energy partitioning approach that takes into account the rate constants of every process involved in the quenching mechanisms of excited chlorophylls. In this study a fully extended quantum yield approach and the introduction of novel equations to assess the yields of each NPQ component are presented. Furthermore, a complete analysis of the yield of NPQ in Beta vulgaris exposed to different irradiances has been carried out. In agreement with experimental results here it is shown that the previous approach may amplify the yield of qE component and flatten the quantitative results of fluorescence analysis. Moreover, the significance of taking into account the physiological variability of NPQ for a correct assessment of energy partitioning is demonstrated.

Investigating The CP29 Photosynthetic Light Harvesting Complex with 2D Electronic Spectroscopy

Though chlorophyll-binding protein CP29, a light harvesting complex in photosystem II of green plants, is proposed to play a role in the regulation of potentially-damaging excess chlorophyll excitations in the supercomplex, little is known about its spatial structure and its relation to excitation energy transfer dynamics and photoprotective action. In vivo, the presence of carotenoid pigment zeaxanthin is correlated with the quenching of excess chlorophyll excitations. Although the mechanism of quenching is still unknown, it is evident that CP29 exchanges carotenoid pigments depending on illumination conditions--in low light, the complex binds violaxanthin, while in high light zeaxanthin is bound.We probe the chlorophyll Qy band of isolated CP29, binding either violaxanthin or zeaxanthin, using conventional and polarization-sensitive two-dimensional electronic spectroscopy (2DES) in order to better characterize electronic and spatial structure. 2DES resolves both excitation and emission energy of the molecular complexes being studied, providing a picture of the correlations between multiple excited states and revealing the presence of states that may go undetected by other spectroscopies. It also provides a direct map of excitation energy transfer processes within the complex by identifying signals from correlated donor and acceptor energies. Polarization-dependent studies provide clues in particular about chromophore configuration. We furthermore investigate whether the binding of zeaxanthin alters the excitation energy landscape and the resulting dynamics of CP29 to potentially modulate the quenching of excess excitations.(Supported by the Office of Basic Energy Sciences, Chemical Sciences Division, U.S. Department of Energy (contract DE-AC03-76S98) and a Seaborg Fellowship to NSG from LBNL).

Delayed fluorescence in photosynthesis

Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon-delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.

Delayed Chlorophyll Fluorescence as a Monitor for Physiological State of Photosynthetic Apparatus

ABSTRACTIntact plants emit light quanta called delayed fluorescence (DF). DF is result of radiative deactivation of secondary excited chlorophyll molecules in Photosystem II (PS II) antennae complexes. The excitations are produced by backward electron- transfer reactions both in the donor and acceptor sides of PS II. The poly-exponential dark decay of DF in a time interval of tens of nanoseconds to tens of seconds reflects the kinetics of different forward and backward reactions of the photosynthetic electron transfer. The current work reviews the mechanisms of the DF light quanta generation and the methodical approaches that allow us to obtain quantitative information about the photosynthetic machinery state using the DF signal from native objects. We examine an approach for the simultaneous record of DF and prompt chlorophyll fluorescence during the transition of the photosynthetic machinery from dark-adapted to light-adapted state. A new device (Senior PEA) built by Hansatech (King's Lynn, UK) allows us to measure simultaneously the induction transients of prompt chlorophyll fluorescence, DF decaying in a time range 10 μs—240 ms, and the changes in transmission at 820 nm. The comparative analysis of the three types of signals and the application of a model-based description of the processes and reactions that determine the dynamics of the signals during the light-induced transitions (dynamic models and JIP-test) allow us to obtain, from a single few-seconds-long measurement, quantitative information for: a) energetic fluxes and efficiencies at different steps of energy transformation; b) rate constants of electron transfer in and between the two photosystems; c) energization of the thylakoid membrane. This illustrates that DF in combination with other optical and luminescent measurements is a highly informative method for investigation of the physiological state of the photosynthetic apparatus of plants in vivo and in situ, and is an indispensable tool for the purposes of the biophysical phenomics.

The Occurrence of the psbS Gene Product in Chlamydomonas reinhardtii and in Other Photosynthetic Organisms and Its Correlation with Energy Quenching†

To avoid photodamage, photosynthetic organisms have developed mechanisms to evade or dissipate excess energy. Lumen overacidification caused by light-induced electron transport triggers quenching of excited chlorophylls and dissipation of excess energy into heat. In higher plants participation of the PsbS protein as the sensor of low lumenal pH was clearly demonstrated. Although light-dependent energy quenching is a property of all photosynthetic organisms, large differences in amplitude and kinetics can be observed thus raising the question whether a single common mechanism is in action. We performed a detailed study of PsbS expression/accumulation in Chlamydomonas reinhardtii and investigated its accumulation in other algae and plants. We showed that PsbS cannot be detected in Chlamydomonas under a wide range of growth conditions. Overexpression of the endogenous psbs gene showed that the corresponding protein could not be addressed to the thylakoid membranes. Survey of different unicellular green algae showed no accumulation of anti-PsbS reactive proteins differently from multicellular species. Nevertheless, some unicellular species exhibit high energy quenching activity, suggesting that a PsbS-independent mechanism is activated. By correlating growth habitat and PsbS accumulation in different species, we suggest that during the evolution the light environment has been a determinant factor for the conservation/loss of the PsbS function.

Inter-pigment interactions in the peridinin chlorophyll protein studied by global and target analysis of time resolved absorption spectra

Inter-pigment interactions define the functioning of light-harvesting protein complexes. To describe the particularly complex molecular dynamics and interactions of peridinin and chlorophyll in the peridinin chlorophyll protein of Amphidinium carterae, we applied global and target analysis to a series of ultrafast transient absorption experiments. We have created and validated a model that consistently describes and characterizes the interactions and evolution of excited and ground-state populations after excitation in all different experiments. The series of energy transfer steps that follow excitation are described by our model of cascading populations and numerous rate constants that correspond to intra-molecular thermal relaxation, fast and slow peridinin-to-chlorophyll energy transfer steps, and chlorophyll excited-state annihilation. By analyzing the spectral response of ground-state peridinins to excited chlorophylls we have identified which specific peridinin molecule is most closely coupled to the chlorophylls. No evidence was found that the intra-molecular charge transfer (ICT) state of peridinin, identified in studies of peridinin in solution, is a separate entity in the protein. The peridinin that exhibited slow peridinin-to-chlorophyll energy transfer was characterized by a difference spectrum free from ICT features, consistent with the importance of coupled ICT and S 1 states for energy transfer.

The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.

Contents of Total Phenolics and Ferulic Acid, and PAL Activity during Water Potential Changes in Leaves of Maize Single‐Cross Hybrids of Different Drought Tolerance

AbstractThe aim of this study was to determine whether the resistance and/or sensitivity to drought stress, can be attributed to the level of phenolic compounds in the leaves of maize genotypes. The experiments were carried out on seedlings of three maize genotypes characterized by different levels of drought resistance. Experiments with three periods of drought were conducted (8, 11 and 14 days), to obtain plants with different levels of water potential in leaves, which induced changes in the total phenolic content and ferulic acid, and l‐phenylalanine ammonia‐lyase (PAL) activity. Only for the drought‐resistant genotype Tina, was the low water potential found to be correlated with the high level of the total phenolic content and ferulic acid, which is the main source of blue fluorescence emissions. Moreover, only for Tina were the highest intensities of blue fluorescence emission correlated with the low water potential in leaves. The phenolic compounds present in leaf tissues can protect the deeper situated mesophyll, by absorbing light reaching the leaf and transforming it into a blue fluorescence. Phenolic compounds can, in this way, function as photoprotectors limiting the excitation of chlorophyll during conditions of water deficit in leaves.

Determination of the excitation migration time in Photosystem II: Consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3 ± 1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~ 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5 ± 0.4 ps) − 1 , the rate of secondary charge separation is (137 ± 5 ps) − 1 and the drop in free energy upon primary charge separation is 826 ± 30 cm − 1 . These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].

Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: How does it divert excess energy from photosynthetic reaction center?

Photosystems must efficiently dissipate absorbed light energy under freezing conditions. To clarify the energy dissipation mechanisms, we examined energy transfer and dissipation dynamics in needles of the evergreen plant Taxus cuspidata by time-resolved fluorescence spectroscopy. In summer and autumn, the energy transfer processes were similar to those reported in other higher plants. However, in winter needles, fluorescence lifetimes became shorter not only in PSII but also in PSI, indicating energy dissipation in winter needles. In addition, almost the same fluorescence spectra were obtained with different excitation wavelengths. In contrast, the fluorescence spectrum showed a large difference due to excitation wavelength in spring needles. The fluorescence spectrum of spring needles in 550-nm excitation showed similar spectra to that of winter needles, however, red-chlorophyll fluorescence was not observed in chlorophyll excitation. These observations suggest that some complexes with some kind of red-shifted carotenoid and red-chlorophyll unlink from the core complex in spring. Seasonal changes of excitation energy dynamics are also discussed in relation to changes in thylakoid stacking.

REACTION OF EXCITED CHLOROPHYLL MOLECULES AT ELECTRODES AND IN PHOTOSYNTHESIS*

Abstract— Excited molecules can exchange electrons with suitable electrodes in an electrochemical cell. Excitation energy of molecules can therefore be converted into electrochemical energy. Electrochemical reactions of excited chlorophyll molecules have been investigated with the help of semiconductor electrodes. It is suggested that this type of reaction also occurs during the primary steps of photosynthesis. Consequently, concepts of electrochemical kinetics would have to be applied in order to elucidate chlorophyll-sensitized reactions in photosynthetic membranes. In order to provide evidence for this conclusion, electrochemical kinetics is applied to the calculation of decay of delayed light from photosynthetic membranes, and the result is compared with experimental data from Chlorella pyrenoidosa. The conversion of light into electrochemical energy via photoelectrochemical reactions of organic dyes in an electrochemical cell is demonstrated, and the postulated analogous electrochemical mechanism for photosynthesis is discussed.

Structurally flexible macro-organization of the pigment–protein complexes of the diatom Phaeodactylum tricornutum

By means of circular dichroism (CD) spectroscopy, we have characterized the organization of the photosynthetic complexes of the diatom Phaeodactylum tricornutum at different levels of structural complexity: in intact cells, isolated thylakoid membranes and purified fucoxanthin chlorophyll protein (FCP) complexes. We found that the CD spectrum of whole cells was dominated by a large band at (+)698 nm, accompanied by a long tail from differential scattering, features typical for psi-type (polymerization or salt-induced) CD. The CD spectrum additionally contained intense (-)679 nm, (+)445 nm and (-)470 nm bands, which were also present in isolated thylakoid membranes and FCPs. While the latter two bands were evidently produced by excitonic interactions, the nature of the (-)679 nm band remained unclear. Electrochromic absorbance changes also revealed the existence of a CD-silent long-wavelength ( approximately 545 nm) absorbing fucoxanthin molecule with very high sensitivity to the transmembrane electrical field. In intact cells the main CD band at (+)698 nm appeared to be associated with the multilamellar organization of the thylakoid membranes. It was sensitive to the osmotic pressure and was selectively diminished at elevated temperatures and was capable of undergoing light-induced reversible changes. In isolated thylakoid membranes, the psi-type CD band, which was lost during the isolation procedure, could be partially restored by addition of Mg-ions, along with the maximum quantum yield and the non-photochemical quenching of singlet excited chlorophyll a, measured by fluorescence transients.

Photosynthesis and non-photochemical excitation quenching components of chlorophyll excitation in maize and field bean during chilling at different photon flux density

The influence of chilling (8 °C, 5 d) at two photon flux densities [PFD, L = 200 and H = 500 μmol(photon) m−2 s−1] on the gas exchange and chlorophyll fluorescence was investigated in chilling-tolerant and chilling-sensitive maize hybrids (Zea mays L., K383×K130, K185×K217) and one cultivar of field bean (Vicia faba L. minor, cv. Nadwiślanski). The net photosynthetic rate (P N) for the both studied plant species was inhibited at 8 °C. P N of both maize hybrids additionally decreased during chilling. Changes in the quantum efficiency of PS2 electron transport (ΦPS2) as a response to chilling and PFD were similar to P N. Measurements of ΦPS2/ΦCO2 ratio showed that in field bean seedlings strong alternative photochemical sinks of energy did not appear during chilling. However, the high increment in ΦPS2/ΦCO2 for maize hybrids can indicate reactions associated with chill damage generation. At 8 °C the non-photochemical quenching (NPQ) increased in all plants with chilling duration and PFD. The appearance of protective (qI,p) and damage (qI,d) components of qI and a decrease in qE (energy dependent quenching) took place. NPQ components of field bean and maize hybrids differed from each other. The amount of protective NPQ (qE + qI,p) components as part of total NPQ was higher in field bean than in maize hybrids at both PFD. On 5th day of chilling, the sum of qE and qI,p was 26.7 % of NPQ in tolerant maize hybrids and 17.6 % of NPQ in the sensitive one (averages for both PFD). The increased PFD inhibited the ability of all plants to perform protective dissipation of absorbed energy. The understanding of the genotypic variation of NPQ components in maize may have implications for the future selection of plants with a high chilling tolerance.

The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light

Young leaves of many plants are transiently red because of the accumulation of anthocyanins, with the redness disappearing as leaves mature. Among the many hypothetical functions of foliar anthocyanins, two are tested in this field study: the sunscreen photoprotective function against excess visible light and the handicap signal against herbivory. We took advantage of intraspecies variation in anthocyanin concentrations of young leaves of Quercus coccifera L. to compare in vivo chlorophyll fluorescence parameters, reflectance spectra, total phenolics and the extent of herbivory of leaves of red- and green-leaved phenotypes occupying the same habitat. Photosystem II (PSII) photochemical efficiencies obtained at various photon fluence rates of red light were similar in green and red leaves. In white light, PSII efficiencies were slightly higher in red leaves than in green leaves, indicating a slight photoprotective role of anthocyanins in the field. However, compared with red phenotypes, green phenotypes suffered greater herbivore damage, as judged by the number of leaves attacked and the area lost to herbivory. In addition, there was a positive correlation between the concentrations of anthocyanins and total phenolics. We suggest that the importance of a photoprotective anthocyanic screen is low in thin, young leaves with low chlorophyll concentrations because the green light attenuated by anthocyanins is less significant for chlorophyll excitation. However, the decreased reflectance in the green spectral band and the concomitant leveling of reflectance throughout the 400-570 nm spectral range may either make red leaves less discernible to some insect herbivores or make insect herbivores more discernible to predators, or both. Moreover, excessive herbivory may be additionally discouraged by the high phenolic concentrations in red leaves.

Inactivation of photosynthetic electron flow during desiccation of desert biological sand crusts and Microcoleus sp.-enriched isolates

Filamentous cyanobacteria, the main primary producers in biological sand crusts, survive harsh environmental conditions including diurnal desiccation/rehydration cycles. Here we describe the inactivation of photosystem II during dehydration of native crusts (NC) and Microcoleus sp. isolates grown on nitrocellulose filters (NCF). The morphology of NCF cells, visualized by scanning-transmission and atomic-force microscopy, disclosed long bacterial filaments encapsulated in extracellular polysaccharides (EPS) tubes consisting of parallel fibrils (100-400 nm wide and 50-100 nm high) oriented mostly perpendicular to the tube length. Presence of empty EPS tubes indicated a gliding capability of the cells. Desiccation of NC resulted in a rapid decline of F(o) and complete loss of F(v). These changes were accompanied by a decrease of 77 K PSII fluorescence emission relative to that of PSI, when excited at 430 nm, and a significant decrease of energy transfer from phycobilisomes to PSII. Lowering the turgor pressure through the addition of 1.5 M trehalose to natural crusts, reduced F(v)/F(m) by over 50% and was accompanied by a decrease of 77 K PSI fluorescence induced by chlorophyll excitation. Excitation of phycobilisomes resulted in a downshift of the PSI emission wavelength by 8 nm, indicative of reduced energy transfer from LHCI to the core PSI. Decline of F(v)/F(m) in trehalose-incubated NCF cells did not induce significant changes in 77 K fluorescence emission. These results suggest that alterations in energy transfer from antennae to reaction centers may be part of the survival strategy of Microcoleus.

Effects of different excitation and detection spectral regions on room temperature chlorophyll a 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 (FV/FM and FV′/FM′), 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 %.

Light-Triggered Action Potentials and Changes in Quantum Efficiency of Photosystem II in Anthoceros Cells

Capillary microelectrodes and pulse amplitude-modulated microfluorometry were used to study light-triggered changes in cell membrane potential, chlorophyll fluorescence, and photochemical yield of PSII in chloroplasts of a hornwort Anthoceros sp. The action potential was generated by illuminating the plant sample for a few seconds. It was accompanied by a reversible decrease in quantum efficiency of PSII and by nonphotochemical quenching of fluorescence that continued as long as 10 min after the light stimulus. The presence of ammonium ions (2 mM) enhanced the amplitude and prolonged the duration of dark changes of fluorescence parameters in accordance with the reported increase in duration and amplitude of the light-triggered action potential in the presence of NH 4 + . A rapid retardation of PSII activity within the first seconds of illumination was also evident from absorbance changes at 810 nm reflecting the redox conversions of chlorophyll P700. The PSII-dependent stage of reduction in the induction curves of P700 absorbance was strongly suppressed, and the amplitudes of signals induced by white and far-red light (717 nm) differed insignificantly. It is concluded that a short-term irradiation triggers the generation of ΔpH at the thylakoid membranes, which is accompanied by inhibition of the plasma membrane H+ pump and by reversible inactivation of PSII due to increased thermal dissipation of chlorophyll excitations.