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Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around -15°C. This, together with the method's easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.
Three methods of algal assays--the standard assay, microassay, and the proposed fluorescence assay--are compared from the point of view of reliability of EC50 detection, the minimum required time for the detection, sensitivity of individual measurement, i.e. at which cell density the particular assay can be used for EC50 estimation, and the time stability of the EC50 values. The assays were performed with green alga Chlorella kessleri strain LARG/1 growing in potassium dichromate solution in Z-medium ranging from 0.01 to 100 mg Cr L⁻¹. The inoculation cell density was set according to the standards to 10⁴ cells mL⁻¹ and according to spectrophotometer/plate reader detection limit. The average EC50 ranged from 0.096 to 0.649 mg Cr L⁻¹ and there were no significant differences in EC50 between the assay type and the inoculation methods with the exception of the significant difference between EC(c)50₇₂ (EC50 established from biomass measured as chlorophyll a concentration after 72 h of cultivation) in the standard assay and EC(r)50 (EC50 derived from growth rate) in the microassay in the standard inoculation experiment due to low variability of their values. The EC(f)50 (EC50 derived from variable fluorescence measurement) values correspond to EC50 values derived from the growth rates. Fluorescence measurement revealed the toxic effect of the chromium after 24 h of exposure at cell density of 5 x 10⁴ cells mL⁻¹, less by half than other used assay methods. The positive correlation of EC(f)50 and time was found in the standard inoculation experiment but opposite effect was observed at the spectrophotometric one.
Lichens survive harsh weather of Antarctica as well as of other hostile environments worldwide. Therefore, this investigation is important to understand the evolution of life on Earth in relation to their stress tolerance strategy. We have used chlorophyll a fluorescence (ChlF) and Raman spectroscopy, respectively, to monitor the activation/deactivation of photosynthesis and carotenoids in three diverse Antarctic lichens, Dermatocarpon polyphyllizum (DP), Umbilicaria antarctica (UA), and Leptogium puberulum (LP). These lichens, post 4 h or 24 h of hydration, showed differences in their ChlF transients and values of major ChlF parameters, e.g., in the maximum quantum efficiency of PSII photochemistry (Fv/Fm), and yields of fluorescence and heat dissipation (Φf,d), of effective quantum efficiency of PSII photochemistry (ΦPSII) and of non-photochemical quenching (Φnpq), which may be due to quantitative and/or qualitative differences in the composition of their photobionts. For understanding the kinetics of hydration-induced activation of photosynthesis, we screened ΦPSII of these lichens and reported its non-linear stimulation on a minute time scale; half of the activation time (t1/2) was fastest ~4.05 ± 0.29 min for DP, which was followed by 5.46 ± 0.18 min for UA, and 13.95 ± 1.24 min for LP. Upon drying of fully activated lichen thallus, there was a slow decay, in hours, of relative water content (RWC) as well as of Fv/Fm. Raman spectral signatures were different for lichens having algal (in DP and UA) and cyanobacteria (in LP) photobionts, and there was a significant shift in ν1(C=C) Raman band of carotenoids post 24 h hydration as compared to their value at a dry state or post 4 h of hydration; this shift was decreased, when drying, in DP and LP but not in UA. We conclude that hydration nonlinearly activated photosynthetic apparatus/reactions of these lichens in minute time range but there was a de-novo synthesis of chlorophylls as well as of carotenoids post 24 h. Their dehydration-induced deactivation, however, was comparatively slow, in hours range, and there seemed a degradation of synthesized chlorophylls and carotenoids post dryness. We conclude that in extremophilic lichens, their photosynthetic partners, in particular, possess a complex survival and photoprotective strategy to be successful in the extreme terrestrial environments in Antarctica.
- MeSH
- Ascomycota MeSH
- chlorofyl a MeSH
- chlorofyl MeSH
- fluorescence MeSH
- fotosyntéza MeSH
- karotenoidy metabolismus MeSH
- lišejníky * metabolismus MeSH
- Ramanova spektroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Antarktida MeSH
Chlorophyll fluorescence kinetic analysis has become an important tool in basic and applied research on plant physiology and agronomy. While early systems recorded the integrated kinetics of a selected spot or plant, later systems enabled imaging of at least the slower parts of the kinetics (20-ms time resolution). For faster events, such as the rise from the basic dark-adapted fluorescence yield to the maximum (OJIP transient), or the fluorescence yield decrease during reoxidation of plastoquinone A after a saturating flash, integrative systems are used because of limiting speed of the available imaging systems. In our new macroscopic and microscopic systems, the OJIP or plastonique A reoxidation fluorescence transients are directly imaged using an ultrafast camera. The advantage of such systems compared to nonimaging measurements is the analysis of heterogeneity of measured parameters, for example between the photosynthetic tissue near the veins and the tissue further away from the veins. Further, in contrast to the pump-and-probe measurement, direct imaging allows for measuring the transition of the plant from the dark-acclimated to a light-acclimated state via a quenching analysis protocol in which every supersaturating flash is coupled to a measurement of the fast fluorescence rise. We show that pump-and-probe measurement of OJIP is prone to artifacts, which are eliminated with the direct measurement. The examples of applications shown here, zinc deficiency and cadmium toxicity, demonstrate that this novel imaging platform can be used for detection and analysis of a range of alterations of the electron flow around PSII.
- MeSH
- Arabidopsis cytologie metabolismus MeSH
- Brassicaceae cytologie účinky léků metabolismus MeSH
- chlorofyl chemie metabolismus MeSH
- design vybavení MeSH
- fluorescence MeSH
- fluorescenční mikroskopie přístrojové vybavení metody MeSH
- fotosyntéza MeSH
- Glycine max cytologie účinky léků metabolismus MeSH
- kinetika MeSH
- listy rostlin cytologie MeSH
- mezofylové buňky metabolismus MeSH
- plastochinon metabolismus MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
The slow kinetic phases of the chlorophyll a fluorescence transient (induction) are valuable tools in studying dynamic regulation of light harvesting, light energy distribution between photosystems, and heat dissipation in photosynthetic organisms. However, the origin of these phases are not yet fully understood. This is especially true in the case of prokaryotic oxygenic photoautotrophs, the cyanobacteria. To understand the origin of the slowest (tens of minutes) kinetic phase, the M-T fluorescence decline, in the context of light acclimation of these globally important microorganisms, we have compared spectrally resolved fluorescence induction data from the wild type Synechocystis sp. PCC 6803 cells, using orange (λ = 593 nm) actinic light, with those of mutants, ΔapcD and ΔOCP, that are unable to perform either state transition or fluorescence quenching by orange carotenoid protein (OCP), respectively. Our results suggest a multiple origin of the M-T decline and reveal a complex interplay of various known regulatory processes in maintaining the redox homeostasis of a cyanobacterial cell. In addition, they lead us to suggest that a new type of regulatory process, operating on the timescale of minutes to hours, is involved in dissipating excess light energy in cyanobacteria.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chlorofyl chemie genetika metabolismus MeSH
- diuron chemie MeSH
- fluorescence MeSH
- fluorescenční spektrometrie MeSH
- fykobilizomy genetika metabolismus MeSH
- kyanid draselný chemie MeSH
- luminiscenční měření MeSH
- světlo MeSH
- Synechocystis chemie genetika metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
One of the elements showing strong beneficial effect on plants at low concentrations and toxic effects at higher concentrations is titanium (Ti). We investigated the interconnection between the Fe uptake and the Ti intoxication in model experiment on Fe-deficient spinach (Spinacia oleracea) plants to help to elucidate the mechanism of the biological activity of titanium in plants. The two different Ti (0 and 20 mg L⁻¹) and two different Fe (0 and 1.35 mg L⁻¹) concentrations in hydroponic medium were used in all four possible combinations. We compared chemical analysis of Ti and Fe in roots and shoots with the changes of the in vivo chlorophyll fluorescence. Although Fe and Ti concentration found in shoots of Ti-non-treated Fe-deficient plants was comparable with that in Ti-treated Fe-deficient plants, the soluble form of Ti present in the growth media had a negative effect on photosynthetic activity monitored by chlorophyll fluorescence measurements. The presence of Fe in growth medium significantly decreased the Ti concentration in shoots and increased the photosynthetic activity. Here, we propose that Ti affect components of electron transport chain containing Fe in their structure (particularly photosystem I) and decrease the photosystem II efficiency.
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
We have used time-resolved absorption and fluorescence spectroscopy with nanosecond resolution to study triplet energy transfer from chlorophylls to carotenoids in a protective process that prevents the formation of reactive singlet oxygen. The light-harvesting complexes studied were isolated from Chromera velia, belonging to a group Alveolata, and Xanthonema debile and Nannochloropsis oceanica, both from Stramenopiles. All three light-harvesting complexes are related to fucoxanthin-chlorophyll protein, but contain only chlorophyll a and no chlorophyll c. In addition, they differ in the carotenoid content. This composition of the complexes allowed us to study the quenching of chlorophyll a triplet states by different carotenoids in a comparable environment. The triplet states of chlorophylls bound to the light-harvesting complexes were quenched by carotenoids with an efficiency close to 100%. Carotenoid triplet states were observed to rise with a ~5 ns lifetime and were spectrally and kinetically homogeneous. The triplet states were formed predominantly on the red-most chlorophylls and were quenched by carotenoids which were further identified or at least spectrally characterized.
- MeSH
- anaerobióza MeSH
- časové faktory MeSH
- chlorofyl metabolismus MeSH
- fluorescenční spektrometrie MeSH
- fotochemické procesy * MeSH
- Heterokontophyta metabolismus MeSH
- karotenoidy metabolismus MeSH
- kinetika MeSH
- proteiny vázající chlorofyl metabolismus MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
