The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.
- Klíčová slova
- auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
- MeSH
- Arabidopsis účinky léků metabolismus ultrastruktura MeSH
- buněčné jádro účinky léků metabolismus ultrastruktura MeSH
- buněčné kultury MeSH
- centrifugace metody MeSH
- frakcionace buněk přístrojové vybavení metody MeSH
- hmotnostní spektrometrie MeSH
- homeostáza fyziologie MeSH
- indoly metabolismus farmakologie MeSH
- kyseliny indoloctové metabolismus MeSH
- protoplasty chemie MeSH
- průtoková cytometrie MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné buňky účinky léků metabolismus ultrastruktura MeSH
- tabák účinky léků metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- indole MeSH Prohlížeč
- indoly MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.
- Klíčová slova
- auxin carriers, correlative microscopy, nanodomains, plasma membrane,
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- buněčná membrána genetika metabolismus ultrastruktura MeSH
- konfokální mikroskopie MeSH
- kovové nanočástice chemie MeSH
- kyseliny indoloctové metabolismus MeSH
- mikroskopie elektronová rastrovací * MeSH
- počítačové zpracování obrazu MeSH
- protoplasty metabolismus ultrastruktura MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- tabák genetika metabolismus ultrastruktura MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
- zlato MeSH
FM (Fei-Mao) styryl dyes are commonly used for the fluorescence imaging of plasma membrane (PM) and endocytosis in vivo. Thanks to their amphiphilic character, these dyes are incorporated in the outer leaflet of the PM lipid bilayer and emit fluorescence in its hydrophobic environment. The endocytic pathway of FM dye uptake starts with rapid PM staining and continues in PM invaginations and membrane vesicles during endocytosis, followed by staining of trans-Golgi network (TGN) and ending in tonoplast (vacuolar membrane). FM dyes do not stain endoplasmic reticulum and nuclear membrane. The time-lapse fluorescence microscopy could track endocytic vesicles and characterize the rate of endocytosis in vivo. On the other hand, fixable FM dyes (FX) can be used for the visualization of particular steps in the FM dye uptake in situ. Staining with FM dyes and subsequent microscopic observations could be performed on both tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and destaining in root tip of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells.
- Klíčová slova
- Arabidopsis thaliana root tip, BY-2, Endocytosis, Endomembranes, FM styryl dyes, Microscopy, Plasma membrane,
- MeSH
- Arabidopsis cytologie ultrastruktura MeSH
- buněčná membrána ultrastruktura MeSH
- endocytóza * MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- kořeny rostlin cytologie ultrastruktura MeSH
- optické zobrazování metody MeSH
- tabák cytologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fluorescenční barviva MeSH
Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.
- MeSH
- buněčná membrána ultrastruktura MeSH
- buněčné jádro ultrastruktura MeSH
- cytoskelet ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- rostlinné buňky ultrastruktura MeSH
- rostliny anatomie a histologie ultrastruktura MeSH
- tabák anatomie a histologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins (CKs) are involved in the regulation of plant development including plastid differentiation and function. Partial location of CK biosynthetic pathways in plastids suggests the importance of CKs for chloroplast development. The impact of genetically modified CK metabolism on endogenous CK, indole-3-acetic acid, and abscisic acid contents in leaves and isolated intact chloroplasts of Nicotiana tabacum was determined by liquid chromatography/mass spectrometry and two-dimensional high-performance liquid chromatography, and alterations in chloroplast ultrastructure by electron microscopy. Ectopic expression of Sho, a gene encoding a Petunia hybrida isopentenyltransferase, was employed to raise CK levels. The increase in CK levels was lower in chloroplasts than in leaves. CK levels were reduced in leaves of tobacco harbouring a CK oxidase/dehydrogenase gene, AtCKX3. The total CK content also decreased in chloroplasts, but CK phosphate levels were higher than in the wild type. In a transformant overexpressing a maize beta-glucosidase gene, Zm-p60.1, naturally targeted to plastids, a decrease of CK-O-glucosides in chloroplasts was found. In leaves, the changes were not significant. CK-O-glucosides accumulated to very high levels in leaves, but not in chloroplasts, of plants overexpressing a ZOG1 gene, encoding trans-zeatin-O-glucosyltransferase from Phaseolus lunatus. Manipulation of the CK content affected levels of indole-3-acetic and abscisic acid. Chloroplasts of plants constitutively overexpressing Sho displayed ultrastructural alterations including the occasional occurrence of crystalloids and an increased number of plastoglobuli. The other transformants did not exhibit any major differences in chloroplast ultrastructure. The results suggest that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of CK metabolism.
- MeSH
- alkyltransferasy a aryltransferasy genetika metabolismus MeSH
- beta-glukosidasa genetika metabolismus MeSH
- chloroplasty metabolismus ultrastruktura MeSH
- cytokininy metabolismus MeSH
- fazol genetika MeSH
- geneticky modifikované rostliny metabolismus ultrastruktura MeSH
- glukosyltransferasy genetika MeSH
- kukuřice setá genetika MeSH
- kyselina abscisová metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- listy rostlin metabolismus ultrastruktura MeSH
- oxidoreduktasy genetika metabolismus MeSH
- Petunia genetika MeSH
- rostlinné proteiny genetika MeSH
- tabák genetika metabolismus ultrastruktura MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenylate isopentenyltransferase MeSH Prohlížeč
- alkyltransferasy a aryltransferasy MeSH
- beta-glukosidasa MeSH
- cytokinin oxidase MeSH Prohlížeč
- cytokininy MeSH
- glukosyltransferasy MeSH
- indoleacetic acid MeSH Prohlížeč
- kyselina abscisová MeSH
- kyseliny indoloctové MeSH
- oxidoreduktasy MeSH
- rostlinné proteiny MeSH
- UDPglucose zeatin O-glucosyltransferase, plant MeSH Prohlížeč
A short review of confocal stereology and three-dimensional image analysis is presented, pointing out the achievements accomplished in this field by the Department of Biomathematics (Institute of Physiology, Prague). One of the methods of confocal stereology, the fakir method for surface area estimation, developed by this laboratory, is described. Methods for automatic measurement of geometrical characteristics of microscopical structures, based on 3-D image processing or surface triangulation, are discussed and compared with interactive stereological methods. Three-dimensional reconstruction programs and software implementation of stereological and digital methods as well as their practical applications are presented. The future trends are discussed.
- MeSH
- choriové klky diagnostické zobrazování MeSH
- konfokální mikroskopie metody MeSH
- lidé MeSH
- matematika MeSH
- počítačové zpracování obrazu MeSH
- povrchové vlastnosti MeSH
- tabák ultrastruktura MeSH
- ultrasonografie MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- srovnávací studie MeSH
Cytokinins play a decisive role in regulation of plastid development and differentiation, but their metabolism in plastids is not known. Metabolic studies using intact chloroplasts are prevented by their instability once they are isolated from leaf cells. Chloroplasts of Nicotiana tabacum L. cv. Petit Havana SR1 were therefore immobilized into low-viscosity alginate. Their intactness was assessed by a glyceraldehyde-3-phosphate dehydrogenase assay which indicated that free chloroplasts totally disintegrated within 7 h, while more than 50% of immobilized chloroplasts remained intact after 24 h. The immobilization had no marked impact on ultrastructure and postponed final destruction. The metabolite profile was similar in free and immobilized chloroplasts after 4 h incubation with tritiated zeatin. Nevertheless, the yield of conversion products decreased twice in immobilized chloroplasts, which was probably the outcome of mass transfer limitations and/or the sorption to polysaccharide matrix.
- MeSH
- buněčné kultury metody MeSH
- chloroplasty fyziologie ultrastruktura MeSH
- cytokininy metabolismus MeSH
- imobilizované buňky fyziologie ultrastruktura MeSH
- kultivované buňky MeSH
- tabák fyziologie ultrastruktura MeSH
- viabilita buněk fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- cytokininy MeSH
- MeSH
- aklimatizace fyziologie MeSH
- cytoplazma fyziologie ultrastruktura MeSH
- cytoskelet fyziologie ultrastruktura MeSH
- intracelulární membrány fyziologie ultrastruktura MeSH
- mikrofilamenta fyziologie ultrastruktura MeSH
- mikrotubuly fyziologie ultrastruktura MeSH
- nízká teplota * MeSH
- organely fyziologie ultrastruktura MeSH
- polymery metabolismus MeSH
- tabák fyziologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- polymery MeSH
A panel of nine antibodies, specific to antigenic determinants located on N- or C-terminal structural domains of alpha and beta subunits of animal tubulin, and antibodies against acetylated, tyrosinated and polyglutamylated tubulins were utilized for probing the Nicotiana tabacum microtubules. The specificity of antibodies was confirmed by immunoblotting on whole cell lysates and on tubulin isoforms separated by high-resolution isoelectric focusing. Whereas antibodies TU-01 and TU-09 reacted with all alpha-tubulin isoforms and TU-06 reacted with all beta-tubulin isoforms, the other antibodies reacted with a limited number of tubulin isoforms. Antibody TU-14 reacted only with two beta-tubulin charge variants. In fixed cells, each of the antibodies stained microtubules of preprophase band, mitotic spindle and phragmoplast. Cortical microtubules were stained by all antibodies except TU-02 and TU-03, which did not decorate microtubules in interphase cells. Immunostaining of unfixed detergent-extracted cells revealed that antibodies against determinants on the C-terminal domains of both subunits decorated microtubules, but these were not stained with antibodies to determinants on the N-terminal domains. These data indicate that in plant microtubules at least several parts of the N-terminal domains of both subunits are either not exposed on the microtubule surface or are masked by the other proteins. In contrast, parts of the C-terminal domains are exposed on the exterior of microtubules. As for animal tubulins the majority of posttranslational modifications as well as binding sites for microtubule-associated proteins (MAPs) have been located to these regions, it is possible also in higher plants that the C-terminal structural domains of both tubulin subunits participate in the modulation of tubulin interactions with associated proteins.
- MeSH
- epitopy MeSH
- fluorescenční mikroskopie MeSH
- jedovaté rostliny * MeSH
- konformace proteinů MeSH
- mikrotubuly metabolismus ultrastruktura MeSH
- molekulární struktura MeSH
- monoklonální protilátky MeSH
- myši MeSH
- prasata MeSH
- tabák metabolismus ultrastruktura MeSH
- tubulin chemie imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- epitopy MeSH
- monoklonální protilátky MeSH
- tubulin MeSH
