The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-μg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
Automated analysis of small and optically variable plant organs, such as grain spikes, is highly demanded in quantitative plant science and breeding. Previous works primarily focused on the detection of prominently visible spikes emerging on the top of the grain plants growing in field conditions. However, accurate and automated analysis of all fully and partially visible spikes in greenhouse images renders a more challenging task, which was rarely addressed in the past. A particular difficulty for image analysis is represented by leaf-covered, occluded but also matured spikes of bushy crop cultivars that can hardly be differentiated from the remaining plant biomass. To address the challenge of automated analysis of arbitrary spike phenotypes in different grain crops and optical setups, here, we performed a comparative investigation of six neural network methods for pattern detection and segmentation in RGB images, including five deep and one shallow neural network. Our experimental results demonstrate that advanced deep learning methods show superior performance, achieving over 90% accuracy by detection and segmentation of spikes in wheat, barley and rye images. However, spike detection in new crop phenotypes can be performed more accurately than segmentation. Furthermore, the detection and segmentation of matured, partially visible and occluded spikes, for which phenotypes substantially deviate from the training set of regular spikes, still represent a challenge to neural network models trained on a limited set of a few hundreds of manually labeled ground truth images. Limitations and further potential improvements of the presented algorithmic frameworks for spike image analysis are discussed. Besides theoretical and experimental investigations, we provide a GUI-based tool (SpikeApp), which shows the application of pre-trained neural networks to fully automate spike detection, segmentation and phenotyping in images of greenhouse-grown plants.
Guard cells on the leaf epidermis regulate stomatal opening for gas exchange between plants and the atmosphere, allowing a balance between photosynthesis and transpiration. Given that guard cells possess several characteristics of sink tissues, their metabolic activities should largely depend on mesophyll-derived sugars. Early biochemical studies revealed sugar uptake into guard cells. However, the transporters that are involved and their relative contribution to guard cell function are not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for glucose import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight mesophyll-derived glucose as an important metabolite connecting stomatal movements with photosynthesis.
The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O2, generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1, we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1. NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1, but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1, but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1, in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O2. In green tissues, this putative effect is masked by photosynthetic O2 evolution. However, O2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.
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
Survival of phototrophic organisms depends on their ability to collect and convert enough light energy to support their metabolism. Phototrophs can extend their absorption cross section by using diverse pigments and by tuning the properties of these pigments via pigment-pigment and pigment-protein interaction. It is well known that some cyanobacteria can grow in heavily shaded habitats by utilizing far-red light harvested with far-red-absorbing chlorophylls d and f. We describe a red-shifted light-harvesting system based on chlorophyll a from a freshwater eustigmatophyte alga Trachydiscus minutus (Eustigmatophyceae, Goniochloridales). A comprehensive characterization of the photosynthetic apparatus of T. minutus is presented. We show that thylakoid membranes of T. minutus contain light-harvesting complexes of several sizes differing in the relative amount of far-red chlorophyll a forms absorbing around 700 nm. The pigment arrangement of the major red-shifted light-harvesting complex is similar to that of the red-shifted antenna of a marine alveolate alga Chromera velia. Evolutionary aspects of the algal far-red light-harvesting complexes are discussed. The presence of these antennas in eustigmatophyte algae opens up new ways to modify organisms of this promising group for effective use of far-red light in mass cultures.
- MeSH
- biologické pigmenty metabolismus MeSH
- diuron MeSH
- fluorescenční spektrometrie MeSH
- Heterokontophyta metabolismus účinky záření MeSH
- membránové proteiny metabolismus MeSH
- sladká voda * MeSH
- světlo * MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- teplota MeSH
- tylakoidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Heat tolerance of plants related to cell membrane thermostability is commonly estimated via the measurement of ion leakage from plant segments after defined heat treatment. To compare heat tolerance of various plants, it is crucial to select suitable heating conditions. This selection is time-consuming and optimizing the conditions for all investigated plants may even be impossible. Another problem of the method is its tendency to overestimate basal heat tolerance. Here we present an improved ion leakage method, which does not suffer from these drawbacks. It is based on gradual heating of plant segments in a water bath or algal suspensions from room temperature up to 70-75°C. The electrical conductivity of the bath/suspension, which is measured continuously during heating, abruptly increases at a certain temperature TCOND (within 55-70°C). The TCOND value can be taken as a measure of cell membrane thermostability, representing the heat tolerance of plants/organisms. Higher TCOND corresponds to higher heat tolerance (basal or acquired) connected to higher thermostability of the cell membrane, as evidenced by the common ion leakage method. The new method also enables determination of the thermostability of photochemical reactions in photosynthetic samples via the simultaneous measurement of Chl fluorescence.
Eutrophication of water by nutrient pollution remains an important environmental issue. The aim of this study was to evaluate the nutrient uptake capacity of an algal biofilm as a means to treat polluted water. In addition, the study investigated the nutrient removal process. The algal biofilm was able to remove 99% of phosphorus within 24 hours of P addition, with the PO4-P concentration in inflowing water ranging from 3 to 10 mg L-1. Different patterns of phosphorus and nitrogen removal were observed. Daily quantity of removed NO3-N ranged from 2 to 25% and was highly dependent on solar irradiance. Precipitation of phosphorus during the removal process was studied using X-ray diffraction analyses and was not confirmed in the biofilm. The biofilm system we constructed has a high efficiency for phosphorus removal and, therefore, has great potential for integration into wastewater treatment processes.
- MeSH
- biodegradace MeSH
- biofilmy * MeSH
- čištění vody * MeSH
- dusík * MeSH
- fosfor * MeSH
- odpad tekutý - odstraňování MeSH
- Publikační typ
- časopisecké články MeSH
An advanced optical biosensor was developed based on the enzymatic reaction with halogenated aliphatic hydrocarbons that is accompanied by the fluorescence change of pH indicator. The device is applicable for the detection of halogenated contaminants in water samples with pH ranging from 4 to 10 and temperature ranging from 5 to 60°C. Main advantages of the developed biosensor are small size (60×30×190mm(3)) and portability, which together with short measurement time of 1min belong to crucial attributes of analytical technique useful for routine environmental monitoring. The biosensor was successfully applied for the detection of several important halogenated pollutants under laboratory conditions, e.g., 1,2-dichloroethane, 1,2,3-trichloropropane and γ-hexachlorocyclohexane, with the limits of detection of 2.7, 1.4 and 12.1mgL(-1), respectively. The continuous monitoring was demonstrated by repetitive injection of halogenated compound into measurement solution. Consequently, field trials under environmental settings were performed. The presence of 1,2-dichloroethane (10mgL(-1)) was proved unambiguously on one of three potentially contaminated sites in Czech Republic, and the same contaminant was monitored on contaminated locality in Serbia. Equipped by Global Positioning System, the biosensor was used for creation of a precise map of contamination. Concentrations determined by biosensor and by gas chromatograph coupled with mass spectrometer exhibited the correlation coefficient of 0.92, providing a good confidence for the routine use of the biosensor system in both field screening and monitoring.
- MeSH
- Bacteria enzymologie MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- chemické látky znečišťující vodu analýza metabolismus MeSH
- chlorované uhlovodíky analýza metabolismus MeSH
- fluorescence MeSH
- fluorescenční spektrometrie přístrojové vybavení MeSH
- hydrolasy metabolismus MeSH
- monitorování životního prostředí přístrojové vybavení MeSH
- voda analýza MeSH
- Publikační typ
- časopisecké články MeSH
The prediction of the world's future energy consumption and global climate change makes it desirable to identify new technologies to replace or augment fossil fuels by environmentally sustainable alternatives. One appealing sustainable energy concept is harvesting solar energy via photosynthesis coupled to conversion of CO2 into chemical feedstock and fuel. In this work, the production of ethylene, the most widely used petrochemical produced exclusively from fossil fuels, in the model cyanobacterium Synechocystis sp. PCC 6803 is studied. A novel instrumentation setup for quantitative monitoring of ethylene production using a combination of flat-panel photobioreactor coupled to a membrane-inlet mass spectrometer is introduced. Carbon partitioning is estimated using a quantitative model of cyanobacterial metabolism. The results show that ethylene is produced under a wide range of light intensities with an optimum at modest irradiances. The results allow production conditions to be optimized in a highly controlled setup.
- MeSH
- autotrofní procesy MeSH
- ethyleny biosyntéza MeSH
- hmotnostní spektrometrie přístrojové vybavení metody MeSH
- kyslík analýza MeSH
- lyasy metabolismus MeSH
- membrány umělé * MeSH
- metabolické sítě a dráhy MeSH
- rekombinace genetická genetika MeSH
- světlo MeSH
- Synechocystis enzymologie růst a vývoj účinky záření MeSH
- uhlík analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
