Isolated Plant Nuclei Research Articles

A protocol to expand plant nuclei.

The resolution achieved by conventional light microscopy is limited by light diffraction. This obstacle can be overcome either by optical super-resolution techniques or by the recently developed method to physically expand specimens, called expansion microscopy (ExM). The method utilizes polymer chemistry and the ability of a swellable polyelectrolyte hydrogel to absorb water, and thus to expand its size. The procedure was successfully applied to different species and tissue samples, mostly from the animal kingdom. Physically expanded nuclei and chromosomes in combination with specific protein labeling and super-resolution microscopy may provide new insight into the ultrastructure, dynamics, and function of plant chromatin. Here we provide a detailed protocol to expand isolated plant nuclei and visualize proteins by indirect immunolabeling. With the focus on chromatin structure, we expanded isolated barley nuclei from root tips and visualized the centromere-specific histone H3 variant CENH3. The achieved physical expansion of ~4.2 times allowed the detection of DAPI-labeled chromatin structures already by conventional wild-field (WF) microscopy with a maximal resolution of ~50-60nm. By applying structured illumination microscopy (SIM), doubling the WF resolution, chromatin structures at a resolution of ~25-35nm were observed. However, a certain distortion of the centromeric chromatin ultrastructure became obvious.

Immunolabeling and In Situ Labeling of Isolated Plant Interphase Nuclei.

Specific labeling of proteins and nucleic acids by immunofluorescence or in situ techniques is an important adjunct to microscopical analysis for cell biology. Labeling of nuclear structures in intact complex tissues is often hampered by problems of penetration of the macromolecular labeling reagents needed. Here we describe a method of labeling isolated plant nuclei that we have found to be a useful approach that can help to overcome these problems.

Autonomous regulation of free Ca 2+ concentrations in isolated plant cell nuclei: A mathematical analysis

Experiments performed on nuclei isolated from animal or plant cells have provided evidence that the nucleus generates directly specific nucleoplasmic calcium transients in response to external stimuli. Recent data suggest that isolated plant nuclei might be considered as a closed system where the nuclear concentration of free calcium would be regulated by reversible movements between the nucleoplasm and nuclear stores. We have addressed the relevance of this hypothesis by developing a mathematical approach to simulate nucleoplasmic calcium dynamics generated under various pH and temperature conditions. Here, we show that the experimental results could be explained provided that calcium channels as well as systems transporting calcium are present on the inner nuclear membrane. The putative channels would allow the entry of calcium into the nucleoplasm whereas the elusive transporting system(s) would contribute to replenish the nuclear stores. The simple proposed model is versatile enough to explain and predict autonomous changes in free calcium in the nucleoplasm of isolated plant nuclei.

Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling.

Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus.

Distribution of gamma-tubulin in cellular compartments of higher plant cells.

Higher plants represent an important group of eukaryotes in which discrete sites for microtubule nucleation are absent in all mitotic and meiotic cells, and a concept of dispersed microtubule-nucleation sites has been generally accepted. Current models of mitotic spindle formation in the absence of centrosomes are based on chromatin-mediated microtubule organization. Moreover, even in cells equipped with centrosomes, microtubules can nucleate on cytoplasmic factors, which are so far poorly characterized (Vorobjev et al., 1997). To understand how microtubules are nucleated and organized without the centrosome, the first step is to determine the molecular composition of the dispersed cytoplasmic or chromatin-associated microtubulenucleation sites. -Tubulin, a distantly related member of the tubulin superfamily, plays a pivotal role in microtubule nucleation. It is organized into the large w2000 kDa open-ring complexes ( -TuRC), located at the centrosomes in animal cells and at the spindle pole bodies in yeast (Moritz et al., 1995). The complete genome of Arabidopsis contains two expressed genes encoding -tubulins. Using affinity purified polyclonal and monoclonal antibodies, directed against different antigenic determinants on the -tubulin molecule, we studied the subcellular distribution of -tubulin in six plant species (Arabidopsis thaliana, Vicia faba, Pisum sativum, Medicago sativa, Hordeum vulgare and Zea mays). Biochemical and immunolocalization studies revealed that -tubulin is highly abundant in different cellular compartments. -Tubulin was localized along all microtubular arrays, discrete staining was found in some interphase nuclei. The size of nuclear spots gradually increased until late G2 when they often occurred as double spots. A similar labelling pattern for -tubulin was found in prekinetochore region of isolated G2 nuclei. -Tubulin was also localized on the nuclear surface, decorating perinuclear microtubules focused to the poles. At the onset of mitosis, when nuclear envelope broke down, -tubulin was found on short kinetochore microtubule fibres organized from kinetochores. A strong signal for -tubulin was associated with kinetochore microtubules along their whole length including the close vicinity of the kinetochores. In cells recovering from anti-microtubular drugs, -tubulin was localized with the re-growing kinetochore microtubules nucleated or captured by kinetochore/centromeric regions. Similarly, on isolated chromosomes, -tubulin co-localized with -tubulin in the kinetochore/centromeric region. Flow cytometry was employed to sort isolated plant nuclei in the G1 and G2 stages of the cell cycle. They were thereafter subjected to quantitative immunoblot analysis. An increase of about 50% of -tubulin in G2 compared to G1 nuclei was found in V. faba. In contrast, when supernatants of centrifuged cell extracts from synchronized cells were analyzed by immunoblotting, there were no differences in the amount of -tubulin between cells in the G1 and G2 stages of the cell cycle (Binarova et al., 2000). Circular configurations of chromosomes with the kinetochore region in the centre and chromosome arms pointing outwards were induced transiently by treatment of root meristems of V. faba, M. sativa and A. thaliana with specific cyclin-dependent protein kinase inhibitors (Binarova et al., 1998a). Kinetochore microtubules, * Corresponding author. Tel. +42-2-4106-2130; fax: +42-4-4106-2384. Cell Biology International 27 (2003) 167–169 Cell Biology International

Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes.

Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and thus must be catalysed by RNA polymerase I. Immunofluorescence and confocal microscopy shows that incorporation occurs in a pattern consisting of many small foci distributed widely through the dense fibrillar component of the nucleoli. Immunogold labelling using silver-enhanced Nanogold probe at the electron microscopic level confirms the sites of transcription as small foci approximately 200 nm in diameter. Simultaneous fluorescence in situ hybridization with a probe to the external transcribed spacer (ETS) region of the pre-rRNA shows that the structures revealed by this probe and the BrUTP immunofluorescence labelling are very similar. A probe to the transcribed portion of the rDNA (18S) also shows a good correlation to the sites of BrUTP incorporation within the nucleolus. On the other hand a probe to the non-transcribed intergenic spacer region (NTS) shows very little coincidence with the sites of BrUTP incorporation, and double fluorescence in situ labelling with both 18S and NTS probes confirms this difference in localization. These results suggest that most BrUTP foci correspond to single transcribed genes.

Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes

SummaryIncorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and thus must be catalysed by RNA polymerase I. Immunofluorescence and confocal microscopy shows that incorporation occurs in a pattern consisting of many small foci distributed widely through the dense fibrillar component of the nucleoli. Immunogold labelling using silver‐enhanced Nanogold probe at the electron microscopic level confirms the sites of transcription as small foci approximately 200nm in diameter. Simultaneous fluorescence in situ hybridization with a probe to the external transcribed spacer (ETS) region of the pre‐rRNA shows that the structures revealed by this probe and the BrUTP immunofluorescence labelling are very similar. A probe to the transcribed portion of the rDNA (18S) also shows a good correlation to the sites of BrUTP incorporation within the nucleolus. On the other hand a probe to the non‐transcribed intergenic spacer region (NTS) shows very little coincidence with the sites of BrUTP incorporation, and double fluorescence in situ labelling with both 18S and NTS probes confirms this difference in localization. These results suggest that most BrUTP foci correspond to single transcribed genes.

Enhancement of the immunocytochemical detection of antigens by microwave irradiation. Benefits and limitations analysed in isolated plant nuclei and Drosophila embryos in toto.

Performing the antibody reaction under controlled heating through microwave irradiation results in significant improvements in the immunovisualization of antigens, such as shortening the times of incubation, lowering the antibody titres used and allowing the detection of difficult, inaccessible antigens. In addition to investigate the basis of the enhancement, we have extended to more intact samples such as isolated plant nuclei and in toto Drosophila embryos the results previously reported only with tissue sections. A drop of heterologous anti-nucleolin antibody covering isolated nuclei from onion root meristems spread on a glass slide was microwave irradiated, resulting in clear immunofluorescent labelling of the nucleoli. This result was never previously obtained in the absence of microwave treatment, even using the complicated procedure previously reported for the homologous identification of this nuclear protein. Using the much larger and to some extent impermeable Drosophila embryos, we were able to show that the incubation time and concentration of the anti-myosin antibody can be strongly reduced by performing the reaction at 45 degrees C under microwave irradiation. The controlled increase in temperature is the main factor responsible for these improvements; the importance of maintaining an adequate mixing of the samples is also emphasized. The proper implementation of these two experimental conditions will require the introduction of appropriate mixing accessories and temperature measuring probes for samples of small volume in current microwave laboratory ovens.

Isolated Plant Nuclei Nucleate Microtubule Assembly: The Nuclear Surface in Higher Plants Has Centrosome-like Activity.

In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucleate MT assembly at a tubulin concentration (14 [mu]M of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxol or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.

Salt effects on total and gene-specific in vitro transcriptional activity of isolated plant nuclei

Nuclei from cell suspension cultures of parsley (Petroselinum crispum L.) were isolated and the effects of salt concentrations on the in vitro transcriptional activity determined. Increasing concentrations of monovalent and divalent cations stimulated α-amanitin sensitive transcriptional activity, whereas α-amanitin insensitive transcriptional activity was not affected. The relative rates of in vitro synthesis of rRNA and four different mRNAs at selected salt concentrations were greatest when the transcription assays were carried out at the lowest salt concentrations tested. Hence, optimal conditions for total RNA synthesis (high salt concentrations) were not necessarily optimal for the run-off transcription of particular genes (low salt concentrations) in isolated nuclei.

Regulation of enzymes in isolated plant nuclei

AbstractPurified nuclei are being used as a test system to study the regulation of nuclear enzymes in plants. Regulatory agents such as light, hormones and polyamines can stimulate kinases or phosphatases that control nuclear protein phosphorylation and they can modulate the activity of as yet unidentified enzymes required for transcript synthesis and/or stabilization. This essay summarizes current findings and discusses the advantages and pitfalls of using isolated nuclei to investigate how nuclear functions are controlled.

The mode of action of chlorsulfuron: The lack of direct inhibition of plant DNA synthesis

Chlorsulfuron (2-chloro- N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzene-sulfonamide), the active ingredient in DuPont Glean Weed Killer, has been proposed to act by inhibiting plant cell division. In order to further define the mode of action of this new herbicide, studies were made of the effects of chlorsulfuron on processes associated with plant DNA synthesis. No inhibitory effects were observed on DNA synthesis in isolated plant nuclei, and the enzymes DNA polymerase and thymidine kinase. Nucleoside precursors of DNA were not effective in lessening chlorsulfuron inhibition of thymidine incorporation into DNA of corn root tips. These results indicate that chlorsulfuron does not inhibit plant cell division by a direct inhibition of DNA synthesis.

Transcription of bacterial DNA by isolated plant nuclei

Plant nuclei prepared from protoplasts can be used as a cell-free system for testing their template activity of procaryotic DNA for plant polymerases. We were able to demonstrate that plant polymerases of Petunia hybrida are capable of transcribing linear bacterial DNA, whereas supercoiled DNA could not be used as a template.

A protein intermediary in the interaction of a hormone with the genome.

The increased rate of RNA synthesis by target cells caused by the plant hormone auxin has been studied as an example of hormonal regulation of transcription. The hormone does not interact directly with chromatin but requires a protein mediator. In the presence of this mediator, auxin increases the rate of RNA synthesis both by isolated plant nuclei and by isolated chromatin. This increased rate of RNA synthesis occurs even in the presence of saturating amounts of RNA polymerase. The hormone and protein do not affect the rate of RNA synthesis if pure DNA is used as the template. The results suggest that auxin plus the protein increase the rate of RNA synthesis by making an increased portion of the genome available for transcription.

Quantitative relationships between deoxyribonucleic and ribonucleic acid in isolated plant nuclei

Nuclei were isolated from the roots of different species of higher plants and the amounts of both deoxyribonucleic acid ( DNA ) and ribonucleic acid ( RNA ) in these nuclei were determined by a modification of the Schmidt-Thannhauser procedure. A new method of isolation was used which involved fixing the roots for a short time in ice-cold 2% (w/v) formaldehyde, and then crushing them in a press. Between 25 and 50% of the nuclei known to be present in the roots could be recovered. It was shown by interferometry, microphotometry and autoradiography, as well as by an examination of all the solutions used, that there was no significant loss of nucleic acids from the nuclei during the isolation procedure. Nuclei from root apices of diploid, tetraploid and hexaploid Allium species, from diploid and tetraploid forms of Tradescantia ohioensis and from Vicia faba , Pisum sativum and Zea mays showed large differences in DNA content. However, these were not accompanied by corresponding differences in RNA content; the DNA ; RNA ratio varied from about unity to more than ten irrespective of the relative amounts of meristematic and differentiating tissue present in the roots. In the nuclei of four of these species, the DNA ; RNA ratio increased as the temperature at which the plants were grown was raised from 4 to 25°C. The results of all these experiments showed the absence of any correlation between the amount of DNA and the total amount of nuclear RNA ; but the possible existence of a correlation between DNA and chromosomal RNA was not excluded.