Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues.

BACKGROUND AND AIMS: The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. METHODS: Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. KEY RESULTS: Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. CONCLUSIONS: The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

Nuclear bodies domain changes with microspore reprogramming to embryogenesis.

We analysed the presence of nuclear bodies and particularly Cajal bodies during representative stages of gametophytic and haploid embryogenic development in isolated microspore and anther cultures of a model system (Brassica napus cv. Topas) and a recalcitrant species (Capsicum annuum L. var. Yolo Wonder B). The nuclear bodies domain is involved on several important roles on nuclear metabolism, and Cajal bodies are specifically involved on the storage and maturation of both snRNPs and snoRNPs, as well as other splicing factors, necessary for mRNA and pre-rRNA processing, but not directly on the transcription. In this study, immunofluorescence and immunogold labelling with anti-trimethylguanosine antibodies against the specific cap of snRNAs, ultrastructural and cytochemical analysis were performed on cryoprocessed samples at confocal and electron microscopy respectively. Results showed that Cajal bodies increase during the early stages of microspore embryogenic development (young pro-embryos), compared to microspore and pollen development. Our results suggest that Cajal bodies may have a role in the transcriptionally active, proliferative stages that characterise early microspore embryogenic development.

Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains.

The immature pollen grain, the microspore, under stress conditions can switch its developmental program towards proliferation and embryogenesis. The comparison between the gametophytic and sporophytic pathways followed by the microspore permitted us to analyse the nuclear changes in plant differentiating cells when switched to proliferation. The nucleus is highly dynamic, the architecture of its well organised functional domains--condensed chromatin, interchromatin region, nuclear bodies and nucleolus--changing in response to DNA replication, RNA transcription, processing and transport. In the present work, the rearrangements of the nuclear domains during the switch to proliferation have been determined by in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling and stress proteins. The study of the changes in the nuclear domains was determined by a correlative approach at confocal and electron microscopy levels. The results showed that the switch of the developmental program and the activation of the proliferative activity affected the functional organization of the nuclear domains, which accordingly changed their architecture and functional state. A redistribution of components, among them various signalling molecules which targeted structures within the interchromatin region upon translocation from the cytoplasm, was also observed.

Changes in extranucleolar transcription during actinomycin D-induced apoptosis.

Actinomycin D (AMD) inhibits DNA-dependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 microg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromo-uridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar RNAs was also affected. Moreover, still in the absence of typical late morphological or biochemical signs of apoptosis (i.e. chromatin condensation), these cells displayed the early apoptotic features, i.e. the externalization of phosphatidylserine residues on the plasma membrane and propidium iodide exclusion in vivo. At the highest concentrations of AMD used, apoptosis massively occurred, with the typical morphological events (progressive chromatin condensation, clustering of snRNPs and SC-35 splicing factor, cell blebbing). However, transcription of hnRNAs was maintained in the residual areas of diffuse chromatin up to advanced apoptotic stages. The inhibition of rRNA synthesis and the defective pre-mRNA maturation seem to be part of the apoptotic process induced by AMD.

New in situ approaches to study the induction of pollen embryogenesis in Capsicum annuum L.

The induction of pollen embryogenesis in Capsicum annuum L. has been studied at the cellular level using various in situ approaches with several molecular probes for DNA, RNA and proteins. The late vacuolated microspore and the young bicellular pollen grain are stages of gametophytic development in which embryogenesis can be induced. Our results show that the late vacuolated microspore stage is most responsive to embryogenesis induction. The proliferating cell nuclear antigen (PCNA) has been immunolocalized at the electron microscopy level, in order to map replication sites in relation to the fine structure of chromatin. It shows different patterns of labelling at both developmental stages studied, revealing that the late vacuolated microspore is in a period of replication. Other in situ studies have been performed to characterize the state of nuclear activity at the specific developmental stages in which the embryogenic induction can occur. The modern in situ terminal-deoxy-nucleotidyl transferase (TdT) reaction for DNA, the immunolocalization of various nuclear antigens (as snRNPs, fibrillarin, RNA) and the ultrastructural in situ hybridization using 18S and 25S ribosomal probes provided valuable data bout the specific features displayed by the functional nuclear compartments of the microspore, and the young vegetative and generative cells. They are related not only to the state of gene activity but also with probably the ability to switch to the sporophytic pathway at specific developmental times of their gametophytic program.

Correlation of nucleolar activity and nucleolar vacuolation in plant cells.

In root meristematic cells nucleolar structure varies with the cell cycle. Apart from normal meristematic nucleoli one finds nucleoli with a big central vacuole surrounded by a loose cortex with individual fibrillar centres [22] clearly visible within it. There are also intermediate structures between both nucleolar types. In Pisum sativum nuclear tissue, the structure of the vacuolated nucleoli is similar and appears in periods of high metabolic activity during megasporogenesis. In both tissues, vacuolated nucleoli incorporate tritiated uridine more actively than 'normal' nucleoli. In this work the structure of spontaneous nucleolar vacuoles is compared with that induced by drugs such as cordycepin, and FUdR. The vacuolated nucleolus with its increased surface corresponds to a transient structure which not only shows higher metabolic activity but also supplies a storing and/or transporting mechanism for nucleolar products.

Characterization of the interchromatin region as the nuclear domain containing snRNPs in plant cells. A cytochemical and immunoelectron microscopy study.

The combination of electron microscopy (EM) cytochemical with immunocytochemical methods is used to characterize the interchromatin region (IR) of the plant cell nucleus. Cryoprocessing of the sample provides a better ultrastructural preservation and allows the observation of some differences in the fine structure of the IR which shows a denser aspect resulting from the lower extraction of components with low-temperature methods. A complex network of fibrillar structures and isolated or clustered 30 to 50-nm granules are observed in the IR. Anti-DNA antibodies combined with the NAMA-Ur method for DNA or the EDTA staining, preferential for RNPs, allow the detection of chromatin fibers in the IR. Bismuth staining reveals the presence of highly phosphorylated proteins in some interchromatin structures. The spliceosomal snRNPs are immunolocalized on cryosections and Lowicryl sections of plant cells using monoclonal and polyclonal antibodies. They provide a homogeneous immunofluorescence pattern with no speckles. This is in correlation with the labeling at EM, immunogold particles decorate the EDTA-positive fibrillar structures of the IR but no labeling is found over the 30 to 50-nm granules. The presence of the spliceosomal snRNPs, DNA and phosphorylated proteins in the IR indicate that this nuclear domain plays a major role in pre-messenger RNA splicing and, possibly in transcription, in the plant cell nucleus.

A new approach to map transcription sites at the ultrastructural level.

We describe a new ultrastructural method for locating transcription on ultra-thin sections. The use of anti-DNA/RNA hybrid antibodies provides specific labeling on precise structures of the nuclear compartments of several cell types. All mammalian and plant material studied (HeLa cells, lymphocytes, onion root meristematic cells) showed the same pattern of labeling: fibrillar structures in the interchromatin region and discrete regions of the dense fibrillar component at the periphery of the fibrillar centers in the nucleolus. The specificity of the immunogold labeling was tested by RNAse H digestion and by pre-blocking the antibody with synthetic DNA/RNA hybrids; in both cases no gold particles were observed. This method has considerable advantages compared with current techniques, constituting a very useful tool to map transcriptionally active loci in a variety of cells.

A specific ultrastructural method to reveal DNA: the NAMA-Ur.

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

Cytochemistry and immunocytochemistry of nucleolar chromatin in plants.

This review attempts to document the most relevant data currently available on the in situ localization of nucleolar chromatin on plant cells. The data provided by the most powerful and recent in situ techniques, such as DNA specific ultrastructural staining, immunogold labelling, in situ molecular cytochemistry, in situ hybridization or confocal microscopy, are summarized and discussed in the light of the potential and limitations of each individual methodology. The presence of DNA in both fibrillar centres and regions of the dense fibrillar component is extensively documented. Data on the nucleolar distribution of other important macromolecules involved in ribosomal transcription are also shown and referred to with regard to the location of DNA. The comparison with the available data on the animal cell nucleolus points towards models of similar functional organization in both plant and animal nucleoli.

Processing of free cells for electron microscopy using a fibrin clot.

A method of fibrin clot preembedding permitting the simple and gentle handling of free cells to be processed for electron microscopy is described. This technique is particularly useful for immunocytochemical techniques such as Lowicryl and thawed croysection approaches and represents a convenient alternative to procedures such as gelatine or agar preembeddings.

Tungsten and molybdenum heteropolyacids as staining and contrasting agents: reactivity with epoxyresin-embedded cell and tissue structures.

In this work, we carry out a further approach to the knowledge of the reaction mechanism of phosphotungstic and phosphomolybdic acids (PTA and PMA), as well as some derivatives, with cell structures from epoxyresin-embedded materials. Applied on thin sections from glutaraldehyde-fixed tissues, PTA and PMA induced a strong electron contrasting reaction in spermatid acrosomes, goblet cell mucin, callose and plant cell walls, endexine, intine and starch granules. In light microscopy, the localization of heteropolyacids on these structures was achieved by treatments of semithin sections with suitable reducing agents (titanous sulfate, stannous chloride, sodium borohydride, or p-phenylenediamine) to form the mixed-valence heteropolyblues, or with Schiffs's reagent. The use of PTA-dye complexes (pyronin-PTA and Mallory's PTA-hematoxylin) also showed the same staining pattern. Taking into account the chemical characteristics of the PTA- and PMA-reactive tissue elements, the present results indicate that heteropolyacids selectively enter into the highest hydrophilic structures from non-polar epoxy-embedded sections; after brief washing, they appear predominantly retained in tissue structures containing a great amount of carbohydrate components.

Visualization of transcription sites at the electron microscope.

In order to localize at EM level the sites of transcription of both pre-mRNA and pre-rRNA, we have detected the DNA/RNA hybrid molecules and m3Gcapped structures by means of specific antibodies after short bromo-uridine (BrU) incorporation. In addition, the sections have been stained by a selective RNA stain, terbium citrate. Our data indicate that perichromatin fibrils incorporate BrU and are labeled by the anti-hybrid probe; this supports the idea that they are the pre-mRNA transcription sites. On the contrary, interchromatin granules do not incorporate BrU after short pulses and are not labeled by the anti-hybrid probe. Concerning the nucleolus, anti-hybrid and anti-BrdU antibodies colocalize only on the dense fibrillar component, suggesting that this is the site of rRNA transcription. Interestingly, the dense fibrillar component and the granular component, after specific RNA staining, show remarkable structural similarities, both containing fibrogranular RNA structures.

A change of developmental program induces the remodeling of the interchromatin domain during microspore embryogenesis in Brassica napus L.

After a stress treatment, in vitro-cultured pollen changes its normal gametophytic developmental pathway towards embryogenesis producing multicellular embryos from which, finally, haploid and double haploid plants develop. The architecture of the well-organized nuclear functional domains changes in response to DNA replication, RNA transcription, processing and transport dynamics. A number of subnuclear structures present in the interchromatin region (IR, the nuclear domain between chromosome territories) have been shown as involved, either directly or indirectly, in transcriptional regulation. These structures include the interchromatin granule clusters (IGCs), perichromatin fibrils (PFs), Cajal bodies (CBs) and perichromatin granules (PGs). In this work, we present a cytochemical, immunocytochemical, quantitative and morphometric analysis at the light, confocal and electron microscopy levels to characterize the changes in the functional architecture of the nuclear interchromatin domain during two developmental programs followed by the microspore: differentiation to mature pollen grains (transcriptionally inactive), and microspore embryogenesis involving proliferation in the first stages (highly engaged in transcription). Our results revealed characteristic changes in size, shape and distribution of the different interchromatin structures as a consequence of the reprogramming of the microspore, allowing us to relate the remodeling of the interchromatin domain to the variations in transcriptional activities during proliferation and differentiation events, and suggesting that RNA-associated structures could be a regulatory mechanism in the process. In addition, we document the presence of two structurally different types of CBs, and of IGC and CB-associated regions, similar to those present in animal cells, and not yet described in plants.

Transmission electron microscopic studies on the mitotic cycle of nucleolar proteins impregnated with silver.

Hela cells were impregnated with silver according to Paweletz et al. (1967). In cells in mitosis not only the nucleolar organizer regions (NORs) are strongly impregnated but also part of the nucleolar material, which accumulates in and around the chromosomes. The treatment with adenosine, which in interphase cells spreads the nucleolar material within the nucleus, also distributes the argentophilic material in and around the chromosomes. During the reconstruction phase this material reassembles around the NORs to form parts of the new nucleolus. The silver impregnation technique clearly demonstrates that two main components are responsible for the argentophily of the nucleolus. This is in agreement with the results obtained by Lischwe et al. (1979).

Ultrastructural nonisotopic mapping of nucleolar transcription sites in onion protoplasts.

The post- and preembedding ultrastructural localization of transcribing rRNA genes has been carried out in nucleoli of permeabilized onion growing root tip protoplasts by means of the nonisotopic bromouridine method. By means of both post- and preembedding approaches, major synthetic sites were identified with morphologically distinct subdomains of dense fibrillar components, with some signal also being associated with nucleolar fibrillar centers and vacuoles. Moreover, labeled medusoid fibrils within distinct domains seen in Lowicryl thin sections likely represent the morphological correlate of transcribing nucleolar genes.

Ultrastructural aspects of tetrads with micronuclei in a wheat monosomic.

Meiosis of monosomics produces tetrad cells with micronuclei. These micronuclei consist exclusively of one chromosome, the critical chromosome. The electron microscope study of these cells reveals that, in the main nucleus the chromatin is relatively uncondensed with large numbers of perichromatin structures and the nucleolus is almost exclusively fibrillar. This corresponds with a phase of active extranucleolar synthesis and moderate nucleolar activity as reported by other authors using biochemical techniques. The chromatin of the micronucleus is in a more condensed stage than in the main nucleus which, in our opinion, is due to a later beginning of interphase. A nuclear envelope surrounds the micronucleus. In its interior, a patch of nucleolar material can be seen. From the available data on the phylogenetic evolution of this special chromosome which constitutes the micronucleus, we suggest that there must be a partial expression of the nucleolar genes carried by this chromosome due to the absence of the factors which impair their expression in disomic plants.

"Ag-NOR" proteins are present when transcription is impaired.

The use of nucleolar silver staining in plant cells in conditions of impaired transcription, either natural (quiescent cells) or induced (actinomycin D, ethidium bromide and cordycepin treatments), shows that nucleoli with very low transcription rates, compared with controls, contain similar amounts of argyrophilic proteins, as well as the same structural location. This demonstrates the absence of correlation between silver staining and present transcriptional activity. The effects of conditions used on the structure of nucleolar chromatin support our previous suggestion on the involvement of "Ag-NOR" proteins in the process of decondensation of NOR chromatin.