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.

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.

MAPKs entry into the nucleus at specific interchromatin domains in plant differentiation and proliferation processes.

Mitogen-activated protein kinases (MAPKs) are involved in the signaling of extracellular stimuli in eukaryotes, including plants. Different MAPKs have recently been shown to be expressed during plant cell proliferation and developmental processes such as pollen development and embryogenesis, but the structural subdomain where these MAPKs are targeted in the nucleus has not yet been characterized. We have determined the changes in the expression and subcellular localization of ERK homologues, proteins belonging to the MAPK family, and MAPK-active forms in two plant developmental processes which involved differentiation (pollen maturation) and proliferation (the initials of pollen embryogenesis). Immunofluorescence and immunogold labeling in the species studied showed that the progression of differentiation and proliferation was accompanied by an increase in the expression of ERKs and MAPK activation together with a translocation to the nucleus. Combining ultrastructural cytochemistry and immunogold for RNA and phosphorylated proteins we have identified the nuclear sites housing these MAPKs in areas of the interchromatin region enriched in RNA and phosphoproteins that include clusters of interchromatin granules. This could suggest a role of these MAPKs in the early events of activation of the transcription and processing machinery, via phosphorylation, which subsequently would be recruited to the transcription sites. The association of the nuclear localization of MAPKs with the progression through the cell cycle and the commitment toward differentiation in the two plant developmental processes can be correlated.

Native and artificial reticuloplasmins co-accumulate in distinct domains of the endoplasmic reticulum and in post-endoplasmic reticulum compartments.

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli.

The rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50-100 RNA polymerases together with the nascent pre-rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as "Christmas trees" 2-3 microm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver-enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to fixed specimens in order to wash out the proteins and RNA, thus specifically revealing DNA strands after uranyl acetate staining. Using this technique, followed by post-embedding in situ hybridization, we observed that the nucleolar rDNA fibres are not extended but show a coiled, thread-like appearance. Our results show for the first time that native rDNA transcription units are linear, compacted Christmas trees.

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development.

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.

The nucleus: a highly organized but dynamic structure.

The nucleus in plants and animals is a highly structured organelle containing several well-defined subregions or suborganelles. These include the nucleolus, interphase chromosome territories and coiled bodies. We have visualized transcription sites in plants at both light- and electron-microscopy level by the incorporation of BrUTP. In the nucleolus many dispersed foci are revealed within the dense fibrillar component, each of which probably corresponds to a single gene copy. In the nucleoplasm there are also many dispersed foci of transcription, but not enough to correspond to one site per transcribed gene. We have shown that in wheat, and probably many other plant species, interphase chromosome territories are organized in a very regular way, with all the chromosomes in the Rabl configuration, all the centromeres clustered at the nuclear membrane and all the telomeres located at the nuclear membrane on the opposite side of the nucleus. However, despite this regular, polarized structure, there is no sign of polarization of transcription sites, or of any preferred location for them with respect to chromosome territorial boundaries. The nucleus is also highly dynamic. As an example, we have shown by the use of a green fluorescent protein fusion to the spliceosomal protein U2B" that coiled bodies move and coalesce within the nucleus, and may act as transport structures within the nucleus and nucleolus.

Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis.

The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program toward a sporophytic pathway, "embryogenesis," has been studied in three different plant species, Brassica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very responsive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes striking structural changes with regard to late gametophytic development, including alterations of biosynthetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after inductive treatment in the nuclei during early embryogenesis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were similar in the three species investigated and represented convenient markers for early monitoring of embryogenesis and selection purposes for obtaining double-haploid plants in plant breeding.

Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains.

Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular stimuli, including mitogens, specific differentiation and developmental signals and stress treatments. This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and yeast, particularly the entry into mitosis of arrested cells. Homologues of MAPKs have been found and cloned in various plant species, but there have been no data about their in situ localization at the subcellular level and their expression in plant cells so far. In the present paper we report the first data on the ultrastructural in situ localization of MAPK and their mRNAs in various plant cells. Proliferating and quiescent meristematic plant cells were studied to evaluate whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells. Moreover, MAPK localization was analyzed in vacuolate microspores. Polyclonal antibodies against the deduced MAPK from the tobacco Ntf6 clone were able to recognize homologue epitopes by immunocytochemical techniques in the cell types studied. The pattern of protein distribution is similar in all the cases studied: it is localized in the cytoplasm and in the nucleus, mainly in the interchromatin region. The quantitative study of the density showed that MAPK labelling is more abundant in cycling than in quiescent cells, also suggesting that, in plants, MAPK pathways might play a role in cell proliferation. RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes were used to study MAPK expression in those plant cells. In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell types studied, but levels were very low in quiescent cells compared to those in cycling cells. The quantification of labelling density of ISH signals strongly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones. Immunogold and ISH results show the presence and distribution of MAPK proteins and mRNAs in vacuolate microspores. This represents a very dynamic stage during pollen development in which the cell nucleus is being prepared for an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain. Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in plant cells.

Histones and DNA ultrastructural distribution in plant cell nucleus: a combination of immunogold and cytochemical methods.

In this work we report for the first time the ultrastructural distribution of histones and DNA in the nuclear compartments in two different plant cell types: Allium cepa L. root meristems and Capsicum annuum L. microspores and pollen grains, by using antibodies against histones H2B and H4 and anti-DNA. Immunolocalizations were combined with ultrastructural cytochemistry for nucleic acids (methylation-acetylation method), DNA (NAMA-Ur) and RNPs (EDTA), to relate the subcellular location of histones and DNA with the chemical subcompartmentalization of the cell nucleus. This is particularly interesting concerning the presence of histones or not on fibers of the interchromatin region and on the fibrillar components of the nucleolus, nuclear subcompartments where transcription has been shown to take place at some regions. Our methodological approach permitted to define precisely the structures where histones were detected in relation to the ultrastructural localization of chromatin in various structural condensation levels. Concerning the localization of DNA and histones on the different components of the nucleolus, the combination of immunogold labeling with the methylation-acetylation cytochemical method, developed in our laboratory, was very useful, thus permitting a clear recognition of the nucleolar components and a correct assignment of labeling, which is not always evident on uranyl-lead-stained Lowicryl sections. Double immunogold assays were also done for a simultaneous visualization of histones and DNA. Our results show a coincident distribution of histones and DNA on the same nuclear compartments revealing the presence of both antigens on condensed chromatin, fibers of the interchromatin region, principally located at the periphery of the condensed chromatin, and in the fibrillar components of the nucleolus.

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.

The immunolocalization of nuclear antigens during the pollen developmental program and the induction of pollen embryogenesis.

The immunolocalization of nuclear antigens, combined with cytochemical procedures as well as in situ hybridization and recent in situ molecular methods, has been applied at different steps of pollen development to characterize the functional organization of the nucleus during the formation of the male gametophyte in an agronomically interesting plant, Capsicum annuum L. Pollen embryogenesis has been induced in pepper and the first stages of the process have been studied at the cellular level. Low temperature processing methods including cryosections and Lowicryl sections were very convenient for performing the various in situ techniques used in the pollen grains. Different molecular probes for localizing DNA, RNA, snRNPs, specific nucleolar proteins, various rRNA species, and DNA/RNA hybrids provided positive results in the pollen nuclei. The data obtained, and the changes observed in the organization of the nuclear compartments during pollen development, are related to the variations in gene activity undergone by the male gametophyte. The methodology used is proposed as a very convenient approach to localize molecules and events involved in the nuclear function in both gametophytic and sporophytic pollen development.

The methylation-acetylation method: an ultrastructural cytochemistry for nucleic acids compatible with immunogold studies.

Methylation-acetylation (MA) is an easy and reproducible ultrastructural cytochemical method which gives preferential contrast to nucleic acid containing structures. When performed en bloc before Lowicryl embedding it does not affect the main antigenic and chemical properties of the sample and is compatible with a large variety of modern immunogold methods permitting a better assignment of the labelling to the well-defined nuclear structures. DNA, RNA, and nuclear proteins, with different chemical nature, nuclear localization, and amount, can be immunolocalized on MA-treated samples. Ultrastructural in situ hybridization and other approaches for studying the functional regions of chromatin, the terminal deoxynucleotidyl transferase and the bromodeoxyuridine methods, are also compatible with the MA procedure. It can be also performed on ultrathin cryosections and Lowicryl sections. A much better visualization of the nuclear structures is obtained, enhancing the distinction between the nucleolar granular and dense fibrillar components. Moreover, the combination of the MA procedure with EDTA regressive staining gives preferential contrast to the RNA-rich structures. It is proposed as a useful approach which can be regularly used for in situ studies of the functional organization of the nucleus in both plant and animal cells.

Immunoelectron microscopy of RNA combined with nucleic acid cytochemistry in plant nucleoli.

The immunoelectron microscopy detection of RNA using anti-RNA monoclonal antibodies has been performed for the first time over different plant cells. The use of the methylation-acetylation (MA) method permits clear distinction among the nuclear and nucleolar compartments and can be combined with the immunogold approach. Cytochemical methods for nucleic acids were performed together with the immunoassays, providing additional data about the different composition of the various nucleolar components. Anti-RNA antibodies highly labeled the ribosome-rich areas of the cytoplasm and the nucleolus. The interchromatin region also is labeled. The labeling was intense in the granular component, lower in the dense fibrillar component, and very scarce in the fibrillar centers. The MA method made possible the statistical evaluation of the labeling density in the various nuclear compartments by permitting the clear assignment of the particles to precise nuclear structures.