Chl Fluorescence Emission Research Articles

Comparative modeling of fluorescence and P700 induction kinetics for alga Scenedesmus sp. obliques and cyanobacterium Synechocystis sp. PCC 6803. Role of state 2-state 1 transitions and redox state of plastoquinone pool.

The model of thylakoid membrane system (T-M model) (Belyaeva et al. Photosynth Res 2019, 140:1-19) has been improved in order to analyze the induction data for dark-adapted samples of algal (Scenedesmus obliques) and cyanobacterial (Synechocystis sp. PCC 6803) cells. The fluorescence induction (FI) curves of Scenedesmus were measured at light exposures of 5 min, while FI and P700 redox transformations of Synechocystis were recorded in parallel for 100 s intervals. Kinetic data comprising the OJIP-SMT fluorescence induction and OABCDEF P700+ absorbance changes were used to study the processes underlying state transitions qT2→1 and qT1→2 associated with the increase/decrease in Chl fluorescence emission. A formula with the Hill kinetics (Ebenhöh et al. Philos Trans R Soc B 2014, 369:20130223) was introduced into the T-M model, with a new variable to imitate the flexible size of antenna AntM(t) associated with PSII. Simulations revealed that the light-harvesting capacity of PSII increases with a corresponding decrease for that of PSI upon the qT2→1 transition induced by plastoquinone (PQ) pool oxidation. The complete T-M model fittings were attained on Scenedesmus or Synechocystis fast waves OJIPS of FI, while SMT wave of FI was reproduced at intervals shorter than 5 min. Also the fast P700 redox transitions (OABC) for Synechocystis were fitted exactly. Reasonable sets of algal and cyanobacterial electron/proton transfer (ET/PT) parameters were found. In the case of Scenedesmus, ET/PT traits remained the same irrespective of modeling with or without qT2→1 transitions. Simulations indicated a high extent (20%) of the PQ pool reduction under dark conditions in Synechocystis compared to 2% in Scenedesmus.

Uptake of diuron and concomitant loss of photosynthetic activity in leaves as visualized by imaging the red chlorophyll fluorescence

The principles of the chlorophyll (Chl) fluorescence induction kinetics (known as Kautsky effect) and their change by the photosystem II herbicide diuron are presented together with the Chl fluorescence emission spectra of a normal and diuron-inhibited leaf. By imaging the Chl fluorescence emission of green leaves the successive uptake of diuron and the concomitant loss of photosynthetic quantum conversion from the leaf base to the leaf tip are documented.

Effects of chlorophyll fluorescence on the estimation of microphytobenthos biomass using spectral reflectance indices

The communities of benthic microalgae that form dense biofilms at the surface of aquatic sediments, or microphytobenthos, are important primary producers in estuarine intertidal flats and shallow coastal waters. The microalgal biomass present in the photic zone of the sediment is a key parameter for ecological and photophysiological studies on microphytobenthos, and has been routinely estimated using hyperspectral reflectance indices based on the chlorophyll (Chl) a red absorption peak at 675 nm, usually the Normalised Difference Vegetation Index (NDVI). This study reports that red region-based biomass indices measured on microphytobenthos biofilms can be significantly affected by the enrichment of reflected light with solar-induced Chl fluorescence emitted by the microalgae. Chl fluorescence emission peaks at 683 nm, counterbalancing the decrease in reflectance centered at 675 nm, thus causing the underestimation of NDVI. The interference of Chl fluorescence was found to be easily identified by a conspicuous double-peak feature in the 670–700 nm region of the second-derivative reflectance spectra. The fluorescence-induced NDVI underestimation was shown to be most pronounced for high surface biomass levels and low incident solar irradiance. Particular aspects of microphytobenthos biofilms, such as the increase in surface Chl fluorescence due the contribution of emission by subsurface layers, and vertical migratory responses by motile microalgae to changes in ambient light, further complicate the effects on biomass estimation using NDVI-like indices. By comparing NDVI with a fluorescence-independent biomass index for a wide range of natural light conditions, it was found that Chl fluorescence interference may cause the underestimation of microalgal biomass to reach over 25%, with errors above 10% being expected for more than half of the measuring occasions. These results indicate that the use of NDVI may compromise the correct assessment of important aspects of microphytobenthos ecology, such as the characterisation of migratory behaviour or the determination of biomass-specific productivity rates, and call for the use of alternative biomass indices, not based on the Chl a red absorption peak.

Impact of an exceptionally hot dry summer on photosynthetic traits in oak (Quercus pubescens) leaves

Climatic constraints on diurnal variations in photosynthetic traits were investigated in oaks (Quercus pubescens Willd.) growing in the Swiss Alps. The measurement period included the summer of 2003, when central Europe experienced a record-breaking heat wave. During the summer, a combination of moderate heat and drought caused a reduction in photosynthetic CO(2) assimilation rate (P(n)) by mid-morning, which increased by the afternoon. More extreme drought and heat caused a sharp day-long reduction in P(n). These effects were closely related to changes in stomatal conductance (g(s)), but low g(s) was unaccompanied by low intercellular CO(2) concentrations (C(i)). Around midday, a combination of heat and drought increased C(i), indicating metabolic limitation of photosynthesis. Chlorophyll a (Chl a) fluorescence measurements revealed reversible down-regulation of photosystem (PS) II activity during the day, which was accentuated by heat and drought and correlated with diurnal variation in zeaxanthin accumulation. A combination of heat and drought reduced leaf Chl a + b concentrations and increased ratios of total carotenoids, xanthophyll-cycle carotenoids and lutein to Chl a + b. The combination of summertime heat and drought altered the 77 K Chl fluorescence emission spectra of leaves, indicating changes in the organization of thylakoid membranes, but it had no effect on the amounts of the major light-harvesting Chl-a/b-binding protein of PSII (LHCII), Rubisco, Rubisco activase, Rubisco-binding protein (cpn-60), phosphoribulokinase and chloroplast ATP synthase. The results demonstrate that Q. pubescens can maintain photosynthetic capacity under adverse summer conditions.

Differential changes in photosynthetic capacity, 77 K chlorophyll fluorescence and chloroplast ultrastructure between Zn‐efficient and Zn‐inefficient rice genotypes (Oryza sativa) under low zinc stress

The relationship of zinc (Zn) efficiency in rice to differential tolerance of photosynthetic capacity and chloroplast function to low Zn stress was studied using Zn-efficient (IR8192) and Zn-inefficient (Erjiufeng) rice genotypes (Oryza sativa L.). Zinc deficiency caused extensive declines in leaf chlorophyll (Chl) content, ratios of chl a:b, Pn, Fv/Fm and Fv/Fo, indicating that the intrinsic quantum efficiency of the photosystem II (PSII) units was damaged. A greater decline was observed in the inefficient genotype (Erjiufeng) than the efficient genotype (IR8192). The 77 K chl fluorescence emission spectrum revealed that Zn deficiency blocked energy spillover from PSII to PSI and more excitation energy was distributed to PSII in IR8192 than Erjiufeng. The spectrum of Zn-deficient Erjiufeng was completely disordered, implying that the photosynthetic centers were seriously damaged. Electron microscopy showed that Zn deficiency caused a severe damage to the fine structure of chloroplasts, but IR8192 had a better preserved chloroplast ultrastructure as compared with Erjiufeng. These differences may result from the higher levels of the antioxidant enzyme activities and lower oxidant stress level in IR8192. These results indicate that Zn deficiency decreases leaf photosynthetic capacity primarily by reducing the number of PSII units per unit leaf area, and also reducing the photochemical capacity of the remaining PSII units. Therefore, the maintenance of more efficient photochemical capacity under low Zn stress is a key factor for the high Zn efficiency in rice, which may result from less antioxidant damage caused by low Zn to the chloroplast ultrastructure.

Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves

Various approaches to understand and make use of the variable chlorophyll (Chl) fluorescence emission spectrum and fluorescence ratio are reviewed. The Chl fluorescence of leaves consists of two maxima in the red (near 685-690 nm), and far-red region (near 730-740 nm). The intensity and shape of the Chl fluorescence emission spectrum of leaves at room temperature are primarily dependent on the concentration of the fluorophore Chl a, and to a lower degree also on the leaf structure, the photosynthetic activity, and the leaf's optical properties. The latter determine the penetration of excitation light into the leaf as well as the emission of Chl fluorescence from different depths of the leaf. Due to the re-absorption mainly of the red Chl fluorescence band emitted inside the leaf, the ratio between the red and the far-red Chl fluorescence maxima (near 690 and 730-740 nm, respectively), e.g., as F690/F735, decreases with increasing Chl content in a curvilinear relationship and is a good inverse indicator of the Chl content of the leaf tissue, e.g., before and after stress events. The Chl fluorescence ratio of leaves can be applied for Chl determinations in basic photosynthesis research, agriculture, horticulture, and forestry. It can be used to assess changes of the photosynthetic apparatus, developmental processes of leaves, state of health, stress events, stress tolerance, and also to detect diseases or N-deficiency of plants.

Heat stress induces an inhibition of excitation energy transfer from phycobilisomes to photosystem II but not to photosystem I in a cyanobacterium Spirulina platensis

The effects of high temperature (30–52.5 °C) on excitation energy transfer from phycobilisomes (PBS) to photosystem I (PSI) and photosystem II (PSII) in a cyanobacterium Spirulina platensis grown at 30 °C were studied by measuring 77 K chlorophyll (Chl) fluorescence emission spectra. Heat stress had a significant effect on 77 K Chl fluorescence emission spectra excited either at 436 or 580 nm. In order to reveal what parts of the photosynthetic apparatus were responsible for the changes in the related Chl fluorescence emission peaks, we fitted the emission spectra by Gaussian components according to the assignments of emission bands to different components of the photosynthetic apparatus. The 643 and 664 nm emissions originate from C-phycocyanin (CPC) and allophycocyanin (APC), respectively. The 685 and 695 nm emissions originate mainly from the core antenna complexes of PSII, CP43 and CP47, respectively. The 725 and 751 nm band is most effectively produced by PSI. There was no significant change in F725 and F751 during heat stress, suggesting that heat stress had no effects on excitation energy transfer from PBS to PSI. On the other hand, heat stress induced an increase in the ratio of Chl fluorescence yield of PBS to PSII, indicating that heat stress inhibits excitation energy transfer from PBS to PSII. However, this inhibition was not associated with an inhibition of excitation energy transfer from CPC to APC since no significant changes in F643 occurred at high temperatures. A dramatic enhancement of F664 occurring at 52.5 °C indicates that excitation energy transfer from APC to the PSII core complexes is suppressed at this temperature, possibly due to the structural changes within the PBS core but not to a detachment of PBS from PSII, resulting in an inhibition of excitation energy transfer from APC to PSII core complexes (CP47 + CP43). A decrease in F685 and F695 in heat-stressed cells with excitation at 436 nm seems to suggest that heat stress did not inhibit excitation energy transfer from the Chl a binding proteins CP47 and CP43 to the PSII reaction center and the decreased Chl fluorescence yields from CP43 and CP47 could be explained by the inhibition of the energy transfer from APC to PSII core complexes (CP47 + CP43).

Differential changes in degradation of chlorophyll–protein complexes of photosystem I and photosystem II during flag leaf senescence of rice

The stability of chlorophyll–protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO 2 assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/ b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll–protein complexes of PSI was greater than in chlorophyll–protein complexes of PSII. The fluorescence yields for all chlorophyll–protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll–protein complex was different and the order for the stability of chlorophyll–protein complexes during leaf senescence was: LHCII > CP43 > CP47 > LHCI > PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll–protein complexes.

Substitution of a Chlorophyll into the Inactive Branch Pheophytin-Binding Site Impairs Charge Separation in Photosystem II

All photosynthetic reaction centers (RCs) have two parallel sets of electron transfer cofactors that cross the membrane. In quinone-type RCs (including photosystem II (PSII)), however, only one pathway (the active branch) is used for electron transfer. Since the electron transfer cofactors of each pathway have nearly identical distance and orientation relationships, it is assumed that local differences in protein environment determine the directionality of electron transfer. To understand further the factors that affect energy distribution among the PSII RC cofactors, we altered the PSII RC cofactor symmetry by replacing the inactive-branch pheophytin (Pheo) with a chlorophyll (Chl). We mutated the D1-L210 residue to a histidine (D1-L210H) to provide a Mg ligand for Chl. Analyses of the pigment composition of D1-L210H RCs indicated that the inactive-branch Pheo had been replaced by a Chl. Comparisons of wild-type and D1-L210 transient absorption spectra confirmed that the red-shifted Pheo Qx absorption band (543.5 nm) belonged to the active-branch Pheo. Surprisingly, intact D1-L210H PSII complexes were unable to evolve oxygen, lacked Chl variable fluorescence, (following a flash), and were unable to photoaccumulate reduced Qa, indicating that electron transfer in D1-L210H PSII complexes was severely perturbed. The kinetics of primary charge separation, however, were not substantially altered in D1-L210H RCs, indicating that the Chl substitution had not perturbed the energetics of the primary electron donor/acceptor pair. Significantly, intact D1-L210H PSII core complexes had a substantially increased and red-shifted Chl fluorescence emission band attributed to fluorescence from Chl's of the distal antenna complex as well as a blue-shifted fluorescence emission peak attributed to Chl's of the proximal antenna complex (77 K). These results are interpreted in terms of a redistribution of the excited-state energy among the pigments of the RC multimer, leading to loss of the excited state via fluorescence in the D1-L210H mutant.

Alteration of Components of Chlorophyll-Protein Complexes and Distribution of Excitation Energy Between the Two Photosystems in Two New Rice Chlorophyll b-less mutants

Two new yellow rice chlorophyll (Chl) b-less (lack) mutants VG28-1 and VG30-5 differ from the other known Chl b-less mutants with larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small sub-unit and smaller amounts of Chl a. We investigated the altered features of Chl-protein complexes and excitation energy distribution in these two mutants, as compared with wild type (WT) rice cv. Zhonghua 11 by using native mild green gel electrophoresis and SDS-PAGE, and 77 K Chl fluorescence in the presence of Mg2+. WT rice revealed five pigment-protein bands and fourteen polypeptides in thylakoid membranes. Two Chl b-less mutants showed only CPI and CPa pigment bands, and contained no 25 and 26 kDa polypeptides, reduced amounts of the 21 kDa polypeptide, but increased quantities of 32, 33, 56, 66, and 19 kDa polypeptides. The enhanced absorption of CPI and CPa and the higher Chl fluorescence emission ratio of F685/F720 were also observed in these mutants. This suggested that the reduction or loss of the antenna LHC1 and LHC2 was compensated by an increment in core component and the capacity to harvest photon energy of photosystem (PS) 1 and PS2, as well as in the fraction of excitation energy distributed to PS2 in the two mutants. 77 K Chl fluorescence spectra of thylakoid membranes showed that the PS1 fluorescence emission was shifted from 730 nm in WT rice to 720 nm in the mutants. The regulation of Mg2+ to excitation energy distribution between the two photosystems was complicated. 10 mM Mg2+ did not affect noticeably the F685/F730 emission ratio of WT thylakoid membranes, but increased the ratio of F685/F720 in the two mutants due to a reduced emission at 685 nm as compared to that at 720 nm.

Quenching of excited states of chlorophyll molecules in submembrane fractions of Photosystem I by exogenous quinones

The ability of three substituted quinones, 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), 2,6-dichloro- p-benzoquinone (DCBQ), and tetramethyl- p-benzoquinone (duriquinone) to quench the excited states of chlorophyll (Chl) molecules in Photosystem I (PSI) was studied. Chl fluorescence emission measured with isolated PSI submembrane fractions was reduced following the addition of exogenous quinones. This quenching progressively increased with rising concentrations of the exogenous quinones according to the Stern–Volmer law. The values of Stern–Volmer quenching coefficients were found to be 3.28×10 5 M −1 (DBMIB), 1.31×10 4 M −1 (DCBQ), and 3.7×10 3 M −1 (duroquinone). The relative quenching capacities of the various exogenous quinones in PSI thus strictly coincided to those found for the quenching of Fo level of Chl fluorescence in isolated thylakoids, which is emitted largely by Photosystem II (PSII) [Biochim. Biophys. Acta (2003) 1604, 115–123]. Quenching of Chl excited states in PSI submembrane fractions by exogenous quinones slowed down the rate of P700, primary electron donor of PSI, photooxidation measured at limiting actinic light irradiances thus revealing a reduced photochemical capacity of absorbed quanta. The possible involvement of non-photochemical quenching of excited Chl states by oxidized phylloquinones, electron acceptors of PSI, and oxidized plastoquinones, mobile electron carriers between PSII and the cytochrome b 6/ f complex, into the control of photochemical activity of PSI is discussed.

Changes in the structure of chlorophyll–protein complexes and excitation energy transfer during photoinhibitory treatment of isolated photosystem I submembrane particles

The activity of light-induced oxygen consumption, absorption spectra, low temperature (77 K) chlorophyll fluorescence emission and excitation spectra were studied in suspensions of photosystem (PS) I submembrane particles illuminated by 2000 μE m −2 s −1 strong white light (WL) at 4 °C. A significant stimulation of oxygen uptake was observed during the first 1–4 h of photoinhibitory treatment, which rapidly decreased during further light exposure. Chlorophyll (Chl) content gradually declined during the exposure of isolated PSI particles to strong light. In addition to the Chl photobleaching, pronounced changes were found in Chl absorption and fluorescence spectra. The position of the major peak in the red part of the absorption spectrum shifted from 680 nm towards shorter wavelengths in the course of strong light exposure. A 6-nm blue shift of that peak was observed after 5-h illumination. Even more pronounced changes were found in the characteristics of Chl fluorescence. The magnitude of the dominating long-wavelength emission band at 736 nm located in untreated particles was five times reduced after 2-h exposure, whereas the loss in absolute Chl contents did not exceed 10% of its initial value. The major peak in low-temperature Chl fluorescence emission spectra shifted from 736 to 721 nm after 6-h WL treatment. Individual Chl–protein complexes differed in the response of their absorption spectra to strong WL. Unlike light-harvesting complexes (LHC), LHCI-680 and LHC-730, which did not exhibit changes in the major peak position, its maximum was shifted from 678 to 671 nm in CPIa complex after PSI submembrane particles were irradiated with strong light for 6 h. The results demonstrated that excitation energy transfer represents the stage of photosynthetic utilization of absorbed quanta which is most sensitive to strong light in isolated PSI particles.

High Irradiance Induced Pigment Degradation and Loss of Photochemical Activity of Wheat Chloroplasts

Loss of chlorophyll (Chl) and carotenoids (Car) of leaves and changes in Chl fluorescence emission and polarisation, malondialdehyde (MDA) accumulation, and 2,6-dichlorophenol indophenol (DCPIP) photoreduction in chloroplasts of wheat seedlings grown under different irradiance and subsequently exposed to high irradiance stress (HIS; 250 W m-2) were studied in mature and senescent primary wheat leaves. Faster rate of pigment loss was observed in leaves of moderate irradiance (MI; 15 W m-2) grown plants, compared to high irradiance (HI-1 and HI-2; 30 and 45 W m-2) ones when exposed to HIS. A relatively lower loss of Car in the plants grown in HI-1 and HI-2 exposed to HIS suggests HI adaptation of these seedlings. The slower rate of increase in the ratio of Chl fluorescence emission (F685/F735) also may suggest photoprotective strategy of HI grown seedlings. There was a positive correlation between MDA accumulation and Chl fluorescence polarisation. The DCPIP photoreduction activity in chloroplasts isolated from HI-1 and HI-2 grown plants exposed to HIS showed slower loss of electron transport activity compared to MI grown plants. These observations suggest that plants grown under higher irradiance have capacity to manage the excess quanta better than those grown under lower irradiance.

Time-resolved chlorophyll fluorescence studies on pigment-protein complexes from photosynthetic membranes

Room-temperature single-photon timing measurements at low excitation energies have been carried out on Photosystem II (PS II) pigment-protein complexes containing 40–70 chlorophylls per reaction centre (RC) as a function of PS II trap closure, Chl fluorescence emission spectra and detergent concentration. The lifetime and amplitude values obtained, using a four exponential model, have been compared with the values exhibited by the isolated light harvesting Chl a b complex of PS II (LHC II) and a Photosystem I (PS I) complex containing 100 Chl/RC. The results suggest that the two rapid decays seen in the PS II preparations arise from the 47 kDa ( т = 54 ps ) and 43 kDa ( т = 180 ps ) Chl containing polypeptides but that in vivo these two proteins give rise to a single variable decay. The differences are discussed in terms of a perturbation in excitation energy migration by detergent molecules, although the presence of high detergent levels does not alter the kinetic and amplitude characteristics of these decays. The two long-lived decays seen in the PS II-core complexes have the same lifetimes and characteristics with respect to detergent addition as the isolated LHC II, suggesting that they arise from LHC II contamination. Unlike the two fast decays, the two long-lived components are extremely sensitive to detergent concentration. The requirement of two decay components in LHC II suggests the presence of a heterogeneous population. Isolated PS I also gives rise to four decay components with 99% of the excitation energy located in pigment-proteins which exhibit lifetimes of 110 ps and 27 ps. The lifetime and amplitude values have been used to calculate estimations for the single-step transfer time between interacting Chl molecules, the number of visits required before trapping takes place and the quantum yield of photochemistry.