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.

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.

Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis.

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7 day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.

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.

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L.

A stress treatment of 32 degrees C for at least 8h was able to change the gametophytic program of the microspore, switching it to embryogenesis in Brassica napus, an interesting model for studying this process in vitro. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores, not sensitive to induction, followed their original gametophytic development. In this work the distribution and ultrastructural localization of two heat-shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins are rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution patterns of these proteins, these changes being assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and noninduced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores, suggesting a possible additional role for Hsp70 in the switch to embryogenesis. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction. The differential appearance and distribution of the two proteins and their association at specific stages have been determined between the two systems coexisting in the same culture: embryogenic development (induced cells) and development of gametes (noninduced cells).

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.

The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division.

We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.

Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus.

The precise location of ribosomal RNA (rRNA) synthesis within the nucleolus is the subject of recent controversy; some investigators have detected nascent RNA in the dense fibrillar components (DFCs) while others have localized transcription to the fibrillar centers (FCs). We endeavored to resolve this controversy by applying a new technique for non-isotopic labeling of RNA and examined the synthesis and movement of non-isotopically labeled rRNA within the nucleolus. We found that rRNA is synthesized only in a restricted area of DFCs, also involving the boundary region with FCs. We traced a movement of RNA from transcription sites through DFCs to granular components. Our results indicate functional compartmentalization of DFCs with respect to the synthesis and processing of precursor rRNA. In situ mapping of the 5' leader sequence of the 5' external transcribed spacer together with transcription labeling indicated that transcription and the first steps in processing of precursor rRNA are spatially separated. Surprisingly, the results also pointed to a partially extended conformation of newly synthesized precursor rRNA transcripts.

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.

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.

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize.

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.

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.

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.

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.

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.