Nowadays there is a great interest in the advancement of the studies on gametic embryogenesis in olive, as a method for obtaining homozygous material, due to the enormous effort and relevance in its genome sequencing and gene mapping. In this study, in vitro isolated microspore culture of four Sicilian olive cultivars ('Biancolilla napoletana', 'Nocellara del Belice', 'Tonda Iblea' and 'Verdello') has been carried out, investigating the influence of two polyamines (putrescine and spermidine) and of two medium compositions on pollen embryogenesis induction. During in vitro development, different structural features have been observed: uninucleated microspores, with no development, binucleated with two asymmetrical nuclei (normal gametophytic pathway: one vegetative and one generative nucleus), binucleated with two equal-size vegetative-type nuclei that had just started their sporophytic pathway, trinucleated, tetranucleated and multinucleated, indicating steps towards the sporophytic development. Moreover, the presence of several calli has been detected. A strong genotype-medium interaction has been observed: each cultivar showed a different response depending on the medium tested.This research represents an advancement in the knowledge about gametic embryogenesis in olive, towards the regeneration of homozygous olive plants.

The current experiments were undertaken to develop a method for regenerating doubled haploid (DH) plants of Eriobotrya japonica (Thunb.), Rosaceae, subfamily Maloideae through isolated microspore culture of nine loquat cultivars. Protocols were developed for isolation and culture of loquat microspores. Isolated microspores in vitro cultured started dividing and developing multicellular, globular and irregular structures. After 4 weeks of culture, the responding microspores produced yellowish/white callus. The embryogenic response of microspores was highly dependent on the cultivar and on the medium composition.

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.

In the recent years, multiple ways of interaction between the fields of nanotechnology and biology have been opened, mainly in the biomedical research, with the development of tools for diagnosis and controlled delivery of substances.1,2 On the other hand, in the field of plant biology, the interaction between both disciplines has been less frequent. Most of the published work on this field has focus in the environmental impact of nanoparticles on crop growth and development;3,4 and also on the bio production of nanoparticles using plant extracts (reviewed in5, as an example see also6,7,8). Much less attention has taken other possible aspects of the interrelationship between nanotechnology and plant biology, such as the development of nanodevices for controlled delivery of drugs or different kind of substances,9,10 in a similar way to that already developed in the medical research.

Haploids and doubled haploids (DHs), very important for their potential use in breeding and genetic analysis, can be induced mainly by two strategies, i.e. by regeneration from the female gamete or from the male gamete. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploidization.In this research, the first stages of the gametic embryogenesis through the anther culture of seven apricot genotypes have been described and characterized through microscopical analysis, carried out at different times of culture.The switch of the developmental microspore programme from the gamethophytic to the sporophytic pathway has been indicated by the presence of multicellular pollen, proembryos and globular embryos in the in vitro system.

Background In recent years, the application of nanotechnology in several fields of bioscience and biomedicine has been studied. The use of nanoparticles for the targeted delivery of substances has been given special attention and is of particular interest in the treatment of plant diseases. In this work both the penetration and the movement of iron-carbon nanoparticles in plant cells have been analyzed in living plants of Cucurbita pepo . Results The nanoparticles were applied in planta using two different application methods, injection and spraying, and magnets were used to retain the particles in movement in specific areas of the plant. The main experimental approach, using correlative light and electron microscopy provided evidence of intracellular localization of nanoparticles and their displacement from the application point. Long range movement of the particles through the plant body was also detected, particles having been found near the magnets used to immobilize and concentrate them. Furthermore, cell response to the nanoparticle presence was detected. Conclusion Nanoparticles were capable of penetrating living plant tissues and migrating to different regions of the plant, although movements over short distances seemed to be favoured. These findings show that the use of carbon coated magnetic particles for directed delivery of substances into plant cells is a feasible application.

Confocal Laser Scanning Microscopy (CLSM) technology and bioimaging are powerful tools for three-dimensional (3D) and colocalization molecular analysis of the microspore embryogenesis. Strategies with fluorescent-labelled probes for in situ hybridization and immunofluorescence have provided unique images of the spatial and temporal pattern of the expression of genes and proteins, and of the sub-cellular rearrangements that accompany the microspore embryogenesis. Various signalling and stress proteins were differentially expressed in reprogrammed microspores and young embryos, and specific endosperm and embryo genes were expressed at different stages, supporting the existence of an endosperm-like domain, in cereals. Specific features such as changes in cell wall components and pectin esterification, presence of callose in special walls, and different behaviour of Cajal nuclear bodies were found in embryogenic microspores and young embryos, constituting early embryogenic markers. The 3D analysis of the nuclear dynamics at early stages of microspore embryogenesis has proved that the nuclear fusion was the mechanism of the spontaneous diploidization.

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.

Root holoparasitic angiosperms, like Orobanche spp, completely lack chlorophyll and totally depend on their host for their supply of nutrients. O. crenata is a severe constraint to the cultivation of legumes and breeding for resistance remains the most economical, feasible, and environmentally friendly method of control. Due to the lack of resistance in commercial pea cultivars, the use of wild relatives for breeding is necessary, and an understanding of the mechanisms underlying host resistance is needed in order to improve screening for resistance in breeding programmes. Compatible and incompatible interactions between O. crenata and pea have been studied using cytochemical procedures. The parasite was stopped in the host cortex before reaching the central cylinder, and accumulation of H2O2, peroxidases, and callose were detected in neighbouring cells. Protein cross-linking in the host cell walls appears as the mechanism of defence, halting penetration of the parasite. In situ hybridization studies have also shown that a peroxidase and a beta-glucanase are differently expressed in cells of the resistant host (Pf651) near the penetration point. The role of these proteins in the resistance to O. crenata is discussed.

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.

Orobanche spp. (broomrapes) are holoparasites lacking in chlorophyll and totally dependent on their host for their supply of nutrients. O. crenata is a severe constraint to legumes cultivation and breeding for resistance remains as one of the best available methods of control. However, little is known about the basis of host resistance to broomrapes. It is a multicomponent event, and resistance based on hampering development and necrosis of broomrape tubercles has been reported. In the present work, the formation of mucilage and occlusion of host xylem vessels associated with the death of O. crenata tubercles were studied histologically. Samples of necrotic O. crenata tubercles established on resistant and susceptible vetch genotypes were collected. The samples were fixed, sectioned and stained using different procedures. The sections were observed at the light microscopy level, either under bright field, epi-fluorescence or confocal laser scanning microscopy. A higher proportion of necrotic tubercles was found on the resistant genotype and this was associated with a higher percentage of occluded vessels. Mucilage is composed mainly by carbohydrates (non-esterified pectins) and the presence of polyphenols was also detected. The mucilage and other substances composed by parasite secretions and host-degraded products was found to block host vessels and obstruct the parasite supply channel, being a quantitative defensive response against O. crenata in vetch, and probably also in other legumes and plants. The presence of foreign substances (i.e. parasite secretions) and host-degraded products (i.e. carbohydrates from cell walls) inside host vessels seems to activate this response and leads to xylem occlusion and further death of established Orobanche tubercles.

ABC Fax + 41 61 306 12 34 E-mail karger@karger.ch www.karger.com © 2005 S. Karger AG, Basel 0301–0171/05/1093–0166$22.00/0 Accessible online at: www.karger.com/cgr Abstract. 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.

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.

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.

Microtubules are made from polymers of alpha/beta dimers. We have observed in rat liver that, on the first day after birth, alpha-subunit is relatively high and beta-subunit low with respect to adult values. In the hypothyroid neonate, both subunits were found to be low, therefore indicating that thyroid hormone (TH) regulates these developmental changes. TH was also found to activate tubulin expression in adult liver, especially beta-subunit. To investigate the role of TH receptors (TRs) in tubulin expression, we analyzed mice lacking TR alpha or TR beta compared with the wild type in both normal and TH-deprived adult animals. The results suggest that, in vivo, beta-tubulin protein expression in the liver is primarily under TR beta positive control. In euthyroid mice lacking TR beta, beta-tubulin expression was low. However, in the corresponding hypothyroid animals, it was found increased, therefore suggesting that the unliganded TR alpha might also upregulate beta-tubulin expression. Accordingly, TH administration to hypothyroid TR beta-deprived mice reduced their high beta-tubulin expression. In parallel, the relatively high messenger level observed with these hypothyroid animals was reduced to the euthyroid level after T-3 treatment. The microtubular network of the mutant livers appeared, by immunofluorescence confocal microscopy, generally disorganized and drastically reduced in beta-tubulin in mice lacking TR beta. In conclusion, our results indicate that beta-tubulin is critically controlled by TR beta in the liver and that both TRs are probably needed to maintain the microtubular network organization of the liver.

The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.

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.

The occurrence and significance of changes in cell wall components and signalling molecules has been investigated during early microspore embryogenesis in cork oak (Quercus suber L.) in relation to cell proliferation and cell differentiation. Microspore embryogenesis has been induced in in vitro anther cultures of Q. suber by the application of a stress treatment of 33 degrees C. After the treatment, microspores at the responsive developmental stage of vacuolate microspore switched towards proliferation and the embryogenesis pathway to further produce haploid plantlets. Ultrastructural and immunocytochemical analysis revealed changes in cell organisation after induction at different developmental stages, the cellular features displayed being in relation to the activation of proliferative activity and the beginning of differentiation in young and late proembryos. Immunogold labelling with JIM5 and JIM7 antibodies showed a different presence of pectin and level of its esterification in cell walls at different developmental stages. Non-esterified pectins were found in higher proportions in cells of late proembryos, suggesting that pectin de-esterification could be related to the beginning of differentiation. The presence and subcellular distribution of Erk 1/2 MAPK homologues have been investigated by immunoblotting, immunofluorescence and immunogold labelling. The results showed an increase in the expression of these proteins with a high presence in the nucleus, during early microspore proembryos development. The reported changes during early microspore embryogenesis are modulated in relation to proliferation and differentiation events. These findings provided new evidences for a role of MAPK signalling pathways in early microspore embryogenesis, specifically in proliferation, and would confer information for the cell fate and the direction of the cell development.