Chl fluorescence Dotaz Zobrazit nápovědu
Photosystem I (PSI) is generally assumed not to emit variable chlorophyll (Chl) fluorescence during light-induced Chl fluorescence rise (FLR), which occurs in a time window upto 1s under high intensity of excitation light. Therefore, the measured FLR and its changes caused by any treatment are usually interpreted by changes only in photosystem II (PSII) fluorescence. But examples can be found in the literature indicating that PSI can emit variable Chl fluorescence at least under certain conditions. As it is impossible to determine the PSI variable Chl fluorescence in vivo solely based on experiments, a way to explore a possible existence of PSI variable Chl fluorescence is to construct a mathematical model of reactions occurring inside and around PSI and to simulate a hypothetical FLR. Based on our present knowledge about the function of PSI, a detailed model describing reactions occurring inside and around PSI was constructed and used for the simulation of FLR originating exclusively in PSI. These simulations show that PSI, in principle, can emit variable Chl fluorescence. Several in silico experiments are performed showing the effect of particular reactions on the FLR. The theoretical PSI variable Chl fluorescence is also compared with theoretical variable fluorescence originating in PSII simulated on the basis of an improved model of PSII showing that variable fluorescence originating in PSI can be as high as 8-17% of overall maximal fluorescence signal originating in both photosystems. The overall FLR obtained as a sum of the simulated FLRs originating in PSI and PSII shows a peak which is similar to an H-peak measured with certain type of samples. We suggest that new experiments be planned to prove the new concept of variable PSI fluorescence.
Quaternary benzo[c]phenanthridine alkaloids (QBAs) are natural products isolated from plants of Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. They are intensively studied for their biological activities, but they have also attractive fluorescence properties. Chromophores responsible for fluorescence are fused aromatic ring systems with electron-donor groups containing oxygen (OH, OCH3, OCH2O). Recently we have described fluorescent characteristics of QBAs - macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) - on interaction with living cells. All these alkaloids immediately enter the living cells and MA-, CHRand SA-bound DNA; they showed a nucleus architecture similar to common DNA dyes. Moreover, MA binds to DNA stoichiometrically and can rapidly report the cellular DNA content in living cells at a resolution adequate for cell cycle analysis. QBAs could be excited by common argon lasers (488 nm) emitting light in the 575-755 nm range. Spectral characteristics of MA allow simultaneous surface immunophenotyping. These characteristics allow multiple applications of the above-mentioned QBAs with significant diagnostic utility. They can be used as supravital fluorescent DNA probes both in fluorescence microscopy and flow cytometry including multiparameter analysis.
Diatoms are especially important microorganisms because they constitute the larger group of microalgae. To survive the constant variations of the light environment, diatoms have developed mechanisms aiming at the dissipation of excess energy, such as the xanthophyll cycle and the non-photochemical chlorophyll (Chl) fluorescence quenching. This contribution is dedicated to the relaxation of the latter process when the adverse conditions cease. An original nonlinear regression analysis of the relaxation of non-photochemical Chl fluorescence quenching, qN, in diatoms is presented. It was used to obtain experimental evidence for the existence of three time-resolved components in the diatom Phaeodactylum tricornutum: qNf, qNi and qNs. qNf (s time-scale) and qNs (h time-scale) are exponential in shape. By contrast, qNi (min time-scale) is of sigmoidal nature and is dominant among the three components. The application of metabolic inhibitors (dithiothreitol, ammonium chloride, cadmium and diphenyleneiodonium chloride) allowed the identification of the mechanisms on which each component mostly relies. qNi is linked to the relaxation of the ΔpH gradient and the reversal of the xanthophyll cycle. qNs quantifies the stage of photoinhibition caused by the high light exposure, qNf seems to reflect fast conformational changes within thylakoid membranes in the vicinity of the photosystem II complexes.
- MeSH
- časové faktory MeSH
- chlorid amonný MeSH
- chlorofyl metabolismus účinky záření MeSH
- dithiothreitol MeSH
- fluorescence MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- kadmium MeSH
- kinetika MeSH
- oniové sloučeniny MeSH
- regresní analýza MeSH
- rozsivky fyziologie MeSH
- světlo * MeSH
- tylakoidy metabolismus MeSH
- xanthofyly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called "deep red fluorescence", a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml-1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the "deep red fluorescence" signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.
- MeSH
- chlorofyl a metabolismus MeSH
- ekosystém MeSH
- fluorescence MeSH
- fotosyntéza MeSH
- fykoerythrin metabolismus MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- oceány a moře MeSH
- Synechococcus chemie klasifikace izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Černé moře MeSH
- oceány a moře MeSH
Response of rice (Oryza sativa) exposed to both biotic and abiotic stresses can be quantified by employing fast and accurate optical methods. In this study, the overall stress responses of (i) 12 near-isogenic lines (NILs) in the genetic background of the rice blast-susceptible cultivar Lijiangxintuanheigu (LTH) and (ii) four NILs in the genetic background of the bacterial blight-susceptible cultivar IR24, were inspected by means of Chl fluorescence (Chl-F) imaging. The distribution of the maximum and effective quantum yield of PSII (Fv/FM and QY) and steady-state Chl-F (Ft) were found to be effective in differentiating symptomatic leaf tissue for both rice blast and bacterial blight, which correlated well with 30 cycles of rice blast and six cycles of bacterial blight previously screened using classical (manual) approaches. Subsequently, identified Chl-F parameters allowing detection under ambient light (QY and Ft) were tested across both biotic and abiotic (drought) stress experiments, for rice cultivars contrasting for drought stress response (N22, IR64 and NSIC Rc 222). Their applicability has been proven for both rice blast and bacterial blight; however, QY failed to detect the effect of drought. In addition to Chl-F, the usefulness of 11 selected vegetation indices (Vis) was tested on these three cultivars exposed to particular stresses: (i) rice blast was detectable by Vis calculated from the visible spectrum; (ii) bacterial blight by near-infrared-related Vis; and (iii) drought by Vis calculated from the visible spectrum. The key Chl-F parameters and/or Vis have been summarized and discussed.
- MeSH
- chlorofyl chemie metabolismus MeSH
- fluorescence MeSH
- fluorometrie MeSH
- fotosystém II - proteinový komplex genetika metabolismus MeSH
- fyziologický stres * MeSH
- interakce hostitele a patogenu MeSH
- listy rostlin genetika metabolismus mikrobiologie MeSH
- Magnaporthe fyziologie MeSH
- nemoci rostlin genetika mikrobiologie MeSH
- období sucha * MeSH
- regulace genové exprese u rostlin MeSH
- rýže (rod) genetika metabolismus mikrobiologie MeSH
- spektrofotometrie MeSH
- Xanthomonas fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Light-harvesting complex II (LHCII) from the marine green macroalga Bryopsis corticulans is spectroscopically characterized to understand the structural and functional changes resulting from adaptation to intertidal environment. LHCII is homologous to its counterpart in land plants but has a different carotenoid and chlorophyll (Chl) composition. This is reflected in the steady-state absorption, fluorescence, linear dichroism, circular dichroism and anisotropic circular dichroism spectra. Time-resolved fluorescence and two-dimensional electronic spectroscopy were used to investigate the consequences of this adaptive change in the pigment composition on the excited-state dynamics. The complex contains additional Chl b spectral forms - absorbing at around 650 nm and 658 nm - and lacks the red-most Chl a forms compared with higher-plant LHCII. Similar to plant LHCII, energy transfer between Chls occurs on timescales from under hundred fs (mainly from Chl b to Chl a) to several picoseconds (mainly between Chl a pools). However, the presence of long-lived, weakly coupled Chl b and Chl a states leads to slower exciton equilibration in LHCII from B. corticulans. The finding demonstrates a trade-off between the enhanced absorption of blue-green light and the excitation migration time. However, the adaptive change does not result in a significant drop in the overall photochemical efficiency of Photosystem II. These results show that LHCII is a robust adaptable system whose spectral properties can be tuned to the environment for optimal light harvesting.
A new method of the chlorophyll (Chl) a fluorescence quenching analysis is described, which allows the calculation of values of (at least) three components of the non-photochemical quenching of the variable Chl a fluorescence (q (N)) using a non-linear regression of a multi-exponential function within experimental data. Formulae for coefficients of the "energy"-dependent (DeltapH-dependent) quenching (q (E)), the state-transition quenching (q (T)) and the photo/inhibitory quenching (q (I)) of Chl a fluorescence were found on the basis of three assumptions: (i) the dark relaxation kinetics of q (N), as well as of all its components, is of an exponential nature, (ii) the superposition principle is valid for individual Chl a fluorescence quenching processes and (iii) the same reference fluorescence level (namely the maximum variable Chl a fluorescence yield in the dark-adapted state, F (V)) is used to define both q (N) and its components. All definitions as well as the algorithms for analytical recognition of the q (N) components are theoretically clarified and experimentally tested. The described theory results in a rather simple equation allowing to compute values for all q (N) components (q (E), q (T), q (I)) as well as the half-times of relaxation (tau(1/2)) of corresponding quenching processes. It is demonstrated that under the above assumptions it holds: q (N) = q (E) + q (T) + q (I). The theoretically derived equations are tested, and the results obtained are discussed for non-stressed and stressed photosynthetically active samples. Semi-empirical formulae for a fast estimation of values of the q (N) components from experimental data are also given.
BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. They can be used as supravital fluorescent DNA probes, both in fluorescence microscopy and flow cytometry, including multiparameter analysis of peripheral blood and bone marrow. MA binds DNA stochiometrically and can provide information on DNA content. Copyright (c) 2007 International Society for Analytical Cytology.
In the major peripheral plant light-harvesting complex LHCII, excitation energy is transferred between chlorophylls along an energetic cascade before it is transmitted further into the photosynthetic assembly to be converted into chemical energy. The efficiency of these energy transfer processes involves a complicated interplay of pigment-protein structural reorganization and protein dynamic disorder, and the system must stay robust within the fluctuating protein environment. The final, lowest energy site has been proposed to exist within a trimeric excitonically coupled chlorophyll (Chl) cluster, comprising Chls a610-a611-a612. We studied an LHCII monomer with a site-specific mutation resulting in the loss of Chls a611and a612, and find that this mutant exhibits two predominant overlapping fluorescence bands. From a combination of bulk measurements, single-molecule fluorescence characterization, and modeling, we propose the two fluorescence bands originate from differing conditions of exciton delocalization and localization realized in the mutant. Disruption of the excitonically coupled terminal emitter Chl trimer results in an increased sensitivity of the excited state energy landscape to the disorder induced by the protein conformations. Consequently, the mutant demonstrates a loss of energy transfer efficiency. On the contrary, in the wild-type complex, the strong resonance coupling and correspondingly high degree of excitation delocalization within the Chls a610-a611-a612 cluster dampens the influence of the environment and ensures optimal communication with neighboring pigments. These results indicate that the terminal emitter trimer is thus an essential design principle for maintaining the efficient light-harvesting function of LHCII in the presence of protein disorder.
Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690 nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S2 and hot S1 states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.
- MeSH
- chlorofyl a * MeSH
- Chlorophyta fyziologie MeSH
- fluorescenční spektrometrie metody MeSH
- Heterokontophyta fyziologie MeSH
- plastidy MeSH
- přenos energie fyziologie MeSH
- Rhodophyta fyziologie MeSH
- světlosběrné proteinové komplexy chemie MeSH
- xanthofyly MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
