Nejvíce citovaný článek - PubMed ID 16228406
Kinetic imaging of chlorophyll fluorescence using modulated light
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
- Názvy látek
- chlorofyl MeSH
- plastochinon MeSH
- zinek MeSH
BACKGROUND: Non-invasive and high-throughput monitoring of drought in plants from its initiation to visible symptoms is essential to quest drought tolerant varieties. Among the existing methods, chlorophyll a fluorescence (ChlF) imaging has the potential to probe systematic changes in photosynthetic reactions; however, prerequisite of dark-adaptation limits its use for high-throughput screening. RESULTS: To improve the throughput monitoring of plants, we have exploited their light-adaptive strategy, and investigated possibilities of measuring ChlF transients under low ambient irradiance. We found that the ChlF transients and associated parameters of two contrasting Arabidopsis thaliana accessions, Rsch and Co, give almost similar information, when measured either after ~20 min dark-adaptation or in the presence of half of the adaptive growth-irradiance. The fluorescence parameters, effective quantum yield of PSII photochemistry (ΦPSII) and fluorescence decrease ratio (RFD) resulting from this approach enabled us to differentiate accessions that is often not possible by well-established dark-adapted fluorescence parameter maximum quantum efficiency of PSII photochemistry (FV/FM). Further, we screened ChlF transients in rosettes of well-watered and drought-stressed six A. thaliana accessions, under half of the adaptive growth-irradiance, without any prior dark-adaptation. Relative water content (RWC) in leaves was also assayed and compared to the ChlF parameters. As expected, the RWC was significantly different in drought-stressed from that in well-watered plants in all the six investigated accessions on day-10 of induced drought; the maximum reduction in the RWC was obtained for Rsch (16%), whereas the minimum reduction was for Co (~7%). Drought induced changes were reflected in several features of ChlF transients; combinatorial images obtained from pattern recognition algorithms, trained on pixels of image sequence, improved the contrast among drought-stressed accessions, and the derived images were well-correlated with their RWC. CONCLUSIONS: We demonstrate here that ChlF transients and associated parameters measured even in the presence of low ambient irradiance preserved its features comparable to that of measured after dark-adaptation and discriminated the accessions having differential geographical origin; further, in combination with combinatorial image analysis tools, these data may be readily employed for early sensing and mapping effects of drought on plant's physiology via easy and fully non-invasive means.
- Klíčová slova
- Chlorophyll fluorescence transients, Drought, Natural accessions, Non-invasive methods, Plant phenotyping, Whole plant rosettes,
- Publikační typ
- časopisecké články MeSH
Trebouxia aggregata (Archibald) Gärtner (phylum Chlorophyta, family Trebouxiaceae), a lichen symbiotic alga, has been identified as host of the well-known herbaceous plant virus Cauliflower mosaic virus (CaMV, family Caulimoviridae). The alga had been isolated from Xanthoria parietina more than 70 years ago and has been maintained in a collection since that time. The CaMV detected in this collection entry has now been completely sequenced. The virus from T. aggregata is mechanically transmissible to a herbaceous host and induces disease symptoms there. Its genome differs by 173 nt from the closest European CaMV-D/H isolate from cauliflower. No site under positive selection was found on the CaMV genome from T. aggregata. We therefore assume that the virus's presence in this alga was not sufficiently long to fix any specific changes in its genome. Apart from this symbiotic alga, CaMV capsid protein sequences were amplified from many other non-symbiotic algae species maintained in a collection (e.g., Oonephris obesa, Elliptochloris sp., Microthamnion kuetzingianum, Chlorella vulgaris, Pseudococcomyxa sp.). CaMV-free Chlorella vulgaris was treated with CaMV to establish virus infection. The virus was still detected there after five passages. The virus infection is morphologically symptomless on Chlorella algae and the photosynthesis activity is slightly decreased in comparison to CaMV-free alga culture. This is the first proof as to the natural presence of CaMV in algae and the first demonstration of algae being artificially infected with this virus.
- MeSH
- Caulimovirus klasifikace genetika izolace a purifikace MeSH
- Chlorophyta virologie MeSH
- DNA virů chemie izolace a purifikace MeSH
- elektronová mikroskopie MeSH
- molekulární sekvence - údaje MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- výročí a významné události MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA virů MeSH
- zlato MeSH
BACKGROUND: An easy and non-invasive method for measuring plant cold tolerance is highly valuable to instigate research targeting breeding of cold tolerant crops. Traditional methods are labor intensive, time-consuming and thereby of limited value for large scale screening. Here, we have tested the capacity of chlorophyll a fluorescence (ChlF) imaging based methods for the first time on intact whole plants and employed advanced statistical classifiers and feature selection rules for finding combinations of images able to discriminate cold tolerant and cold sensitive plants. RESULTS: ChlF emission from intact whole plant rosettes of nine Arabidopsis thaliana accessions was measured for (1) non-acclimated (NAC, six week old plants grown at room temperature), (2) cold acclimated (AC, NAC plants acclimated at 4°C for two weeks), and (3) sub-zero temperature (ST) treated (STT, AC plants treated at -4°C for 8 h in dark) states. Cold acclimation broadened the slow phase of ChlF transients in cold sensitive (Co, C24, Can and Cvi) A. thaliana accessions. Similar broadening in the slow phase of ChlF transients was observed in cold tolerant (Col, Rsch, and Te) plants following ST treatments. ChlF parameters: maximum quantum yield of PSII photochemistry (FV/FM) and fluorescence decrease ratio (RFD) well categorized the cold sensitive and tolerant plants when measured in STT state. We trained a range of statistical classifiers with the sequence of captured ChlF images and selected a high performing quadratic discriminant classifier (QDC) in combination with sequential forward floating selection (SFFS) feature selection methods and found that linear combination of three images showed a reasonable contrast between cold sensitive and tolerant A. thaliana accessions for AC as well as for STT states. CONCLUSIONS: ChlF transients measured for an intact whole plant is important for understanding the impact of cold acclimation on photosynthetic processes. Combinatorial imaging combined with statistical classifiers and feature selection methods worked well for the screening of cold tolerance without exposing plants to sub-zero temperatures. This opens up new possibilities for high-throughput monitoring of whole plants cold tolerance via easy and fully non-invasive means.
- Klíčová slova
- Arabidopsis thaliana, Chlorophyll a fluorescence transients, Cold acclimation, Cold tolerance, High-throughput screening, Whole plant,
- Publikační typ
- časopisecké články MeSH
The acidophilic red alga Cyanidium sp. is one of the dominant mat-forming species in the highly acidic waters of Río Tinto, Spain. The culture of Cyanidium sp., isolated from a microbial mat sample collected at Río Tinto, was exposed to 9 different pH conditions in a gradient from 0.5 to 5 for 24 h and its physiological status evaluated by variable chlorophyll a fluorescence kinetics measurements. Maximum quantum yield was determined after 30 min, 1 h, 2 h, 4 h, 6 h and 24 h of exposure after 15 min dark adaptation. The effect of pH on photochemical activity of Cyanidium sp. was observable as early as 30 min after exposure and the pattern remained stable or with only minor modifications for 24 h. The optimum pH ranged from 1.5 to 2.5. A steep decrease of the photochemical activity was observed at pH below 1 even after 30 min of exposure. Although the alga had tolerated the exposure to pH = 1 for at least 6 h, longer (24 h) exposure resulted in reduction of the photochemical activity. At pH above 2.5, the decline was more moderate and its negative effect on photochemistry was less severe. According to the fluorescence measurements, the red alga Cyanidium sp. is well-adapted to prevailing pH at its original locality at Río Tinto, i.e. pH of 1 to 3. The short-term survival in pH < 1.5 may be adaptation to rare exposures to such low pH in the field. The tolerance of pH above 3 could be caused by adaptation to the microenvironment of the inner parts of microbial mats in which Cyanidium sp. usually dominates and where higher pH could occur due to photosynthetic oxygen production.
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.
- MeSH
- aklimatizace * MeSH
- Arabidopsis fyziologie MeSH
- chlorofyl fyziologie MeSH
- elektrolyty metabolismus MeSH
- fluorescence MeSH
- fyziologický stres MeSH
- nízká teplota * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chlorofyl MeSH
- elektrolyty MeSH
Automatic discrimination of plant species is required for precision farming and for advanced environmental protection. For this task, reflected sunlight has already been tested whereas fluorescence emission has been only scarcely considered. Here, we investigated the discriminative potential of chlorophyll fluorescence imaging in a case study using three closely related plant species of the family Lamiaceae. We compared discriminative potential of eight classifiers and four feature selection methods to identify the fluorescence parameters that can yield the highest contrast between the species. Three plant species: Ocimum basilicum, Origanum majorana and Origanum vulgare were grown separately as well as in pots where all three species were mixed. First, eight statistical classifiers were applied and tested in simulated species discrimination. The performance of the Quadratic Discriminant Classifier was found to be the most efficient. This classifier was further applied in combination with four different methods of feature selection. The Sequential Forward Floating Selection was found as the most efficient method for selecting the best performing subset of fluorescence images. The ability of the combinatorial statistical techniques for discriminating the species was also compared to the resolving power of conventional fluorescence parameters and found to be more efficient.
Localized infection of a plant can be mapped by a sequence of images capturing chlorophyll fluorescence transients in actinic light. Choice of the actinic light protocol co-determines fluorescence contrast between infected leaf segment and surrounding healthy tissue. Frequently, biology cannot predict with which irradiance protocol, in which fluorescence image of the sequence, and in which segment of the image there will be the highest contrast between the healthy and infected tissue. Here, we introduce a new technique that can be applied to identify the combination of chlorophyll fluorescence images yielding the highest contrast. The sets of the most contrasting images vary throughout the progress of the infection. Such specific image sets, stress-revealing fluorescence signatures, can be found for the initial and late phases of the infection. Using these signatures, images can be divided into segments that show tissue in different infection phases. We demonstrate the capacity of the algorithm in an investigation of infection of the model plant Arabidopsis thaliana by the bacterium Pseudomonas syringae. We show that the highest contrast is found with transients elicited by fluctuating, harmonically modulated irradiance with long periods.
Apples were exposed to various concentrations of roseotoxins - metabolites of Trichothecium roseum and kinetic fluorescence imaging was used to detect the area influenced by the phytotoxin. Contrast was quantified within these images between the areas exposed to roseotoxins and the untreated areas. It was proved that roseotoxin B is able to penetrate apple peel and produce chlorotic lesions. Activity of roseotoxin B is similar as the activity of destruxins, host specific phytotoxins of Alternaria brassicae parasitic on canola.
Plants exposed to harmonically modulated irradiance, approximately 1 + cos(omegat), exhibit a complex periodic pattern of chlorophyll fluorescence emission that can be deconvoluted into a steady-state component, a component that is modulated with the frequency of the irradiance (omega), and into at least two upper harmonic components (2omega and 3omega). A model is proposed that accounts for the upper harmonics in fluorescence emission by nonlinear negative feedback regulation of photosynthesis. In contrast to simpler linear models, the model predicts that the steady-state fluorescence component will depend on the frequency of light modulation, and that amplitudes of all fluorescence components will exhibit resonance peak(s) when the irradiance frequency is tuned to an internal frequency of a regulatory component. The experiments confirmed that the upper harmonic components appear and exhibit distinct resonant peaks. The frequency of autonomous oscillations observed earlier upon an abrupt increase in CO(2) concentration corresponds to the sharpest of the resonant peaks of the forced oscillations. We propose that the underlying principles are general for a wide spectrum of negative-feedback regulatory mechanisms. The analysis by forced harmonic oscillations will enable us to examine internal dynamics of regulatory processes that have not been accessible to noninvasive fluorescence monitoring to date.
- MeSH
- biofyzika MeSH
- biofyzikální jevy MeSH
- chlorofyl MeSH
- fluorescenční spektrometrie MeSH
- Fourierova analýza MeSH
- kinetika MeSH
- listy rostlin metabolismus MeSH
- oscilometrie * MeSH
- oxid uhličitý MeSH
- rostliny chemie metabolismus MeSH
- spektrofotometrie infračervená MeSH
- statistické modely MeSH
- statistika jako téma MeSH
- tabák metabolismus MeSH
- videomikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl MeSH
- oxid uhličitý MeSH
