Monica Ruffini Castiglione

Molecular geometry of antigen binding by a monoclonal antibody against 5-methylcytidine

1. The specificity of a monoclonal IgG1 raised against a 5-methylcytidine-keyhole limpet hemocyanin conjugate was investigated by inhibition experiments with soluble competing antigens. 2. A competitive enzyme immunoassay has been set up, with the antigen immobilized on polystyrene microtitration wells. 3. The analysis of the cross-reaction profile allowed the topography of the antigen-antibody interaction to be described. 4. The binding properties of the monoclonal antibody are discussed in terms of both analytical applications and working limitations in the immunochemical study of gene methylation.

Arsenic-induced morphogenic response in roots of arsenic hyperaccumulator fern Pteris vittata

On the assumption that arsenic induces stress morphogenetic responses involved in As tolerance and hyperaccumulation in the Pteris vittata fern, we analyzed the root system of young sporophytes grown in 250, 334, and 500 μM As for five days and for 14 days. Anatomical and histological analyses were performed in plants grown for five days to evaluate the number, position, length and differentiation pattern of root hairs. AgNOR staining, employed to study nucleolus behavior in root apices, showed that arsenic influences nucleolar activity (evaluated by nucleolus size, number and absorbance) in the root meristem. In plants treated with 250 and 334 μM As an acropetal shift of root hair development and an increase in hair length and density were observed, linked to an ectopic pattern of differentiation. The opposite trend was recorded in plants treated with 500 μM As. It is worth noting the presence of living border-like cells, not yet observed in ferns, and their increase following As treatments. Analysis and vitality of border-like cells were surveyed after 14 days of treatments. In conclusion As treatments elicited a stress-induced morphogenic response which, by modifying the differentiation pattern, number and length of root hairs, modulating nucleolar activity and interacting with the rhizosphere by inducing border-like cell production, may adjust the rate of root uptake and its metabolic activity.

A fascinating island: 2n = 4

Abstract Among Angiosperms, only six species are known to possess the lowest identified chromosome number, i.e. 2n = 4. These plants are the monocotyledons Zingeria biebersteiniana, Colpodium versicolor, Ornithogalum tenuifolium and Rhynchospora tenuis, and the dicotyledons Haplopappus gracilis and Brachyscome dichromosomatica. The low chromosome number may be cytogenetically derived from a different ancestral basic number, characteristic of each genus, by different processes, including tandem fusion or unequal reciprocal translocations with loss of centric fragments. All these plants possess low nuclear 4C DNA contents, ranging from 1.56 pg (R. tenuis) to 8.20 pg (H. gracilis), and they generally display a similar chromosome size and a similar position of the nucleolus organising region (NOR), that is often located in the terminal or subterminal region of the small chromosome pair. All these characteristics could be a consequence of common adaptative mechanisms. Peculiar characteristics within these karyotypes are the holocentric chromosomes of R. tenuis and the presence of B chromosomes in B. dichromosomatica. Plants with a very low chromosome number may be considered to constitute a fascinating “island of interest”; moreover, they represent simple systems helpful for the examination of the structural organisation and evolution of Angiosperm chromosomes.

Cytogenetic and histological approach for early detection of "mantled" somaclonal variants of oil palm regenerated by somatic embryogenesis: first results on the characterization of regeneration system.

Abstract The occurrence of anomalous cytological events during the cell proliferation of plant primary explants, and the prevalence of polyploidization and chromosome reduction events, mainly induced by in vitro culture stress, generate large genome variability among cultured committed cells and successively regenerated plants. A thorough cytological analysis on different embryogenic and non-embryogenic oil palm (Elaeis guineensis Jacq.) calli was performed, with the aim to identifying chromosome instability as a possible cause of soma-clonal variants in the regenerated plants. Besides, the process of embryoids formation from embryogenic calli was dissected, setting up a fast and reliable system of tissue culture from zygotic embryos. Our results demonstrated that in the cultured explants of oil palm, similarly to other plant species, the initial events leading to embryogenic/regeneration commitment were managed by a reprogramming of somatic cells towards a gamete-like state, including chromosomes segregation and the occurrence of haploid gamete-like cells. The intrinsic instability of oil palm chromosomes was also verified.

Methylated DNA sequence extrusion during plant early meiotic prophase

Abstract Reproduction in plants has been thoroughly studied and abundant information is currently available in scientific literature. Here we present new data related to a process of DNA modulation along the meiotic steps, first described 50 years ago, but never further pursued. A time course of DNA content/nucleus by cytophotometric analysis, along the reproductive steps from Pollen Mother Cells (PMCs) to seedlings in barley and oil palm displays a significant modulation of nuclear DNA content that reaches its minimum at the zygote stage. Recovery of the DNA content/nucleus during seed germination stages has been demonstrated. Moreover, cytological and immunocytochemical studies indicate that methylated heterochromatic bodies are extruded from PMC nuclei in the progression of the pre-leptotene stage up to the bouquet. The heterochromatic and methylated status of the extruded bodies gives these data a perspective aligned with exciting current data relating to DNA methylation dynamics and chromatin modifications. Our hypothesis is that somatic cells committed to undergoing meiosis need to release the burden of accumulated somatic multicopy DNA sequences and regain totipotency before entering the process of gametogenesis. The event is interpreted as a mechanism for assuring a single copy DNA pairing and therefore avoiding the risk of unequal crossing-over. Methylation represents the evidence of RNA interference machinery at work, leading to heterochromatinization and extrusion of sequences. The recovery of the extruded DNA sequences along embryo development suggests the occurrence of gene amplification mechanisms.

Methylated-rich regions and tandem repeat arrays along the chromosome complement of Colpodium versicolor (Stev.) Schmalh.

The grass Colpodium versicolor (Stev.) Schmalh is one of six angiosperms with extremely reduced chromosome set 2n = 2x = 4. The chromosome complement of this species was studied. The DNA methylation pattern was determined with a specific monoclonal antiboby. 5-Methylcytosine residues are present in different chromosomal sites, with specific occurrence, some methylated bands showing differences between homologous chromosomes. Moreover, a fluorescent in situ hybridisation with telomere repeats and 45S rDNA sequences were performed. Hybridisation signals of telomeric repeats are detectable at the distal ends of the two pair of chromosomes, while 45S rDNA is localised in one chromosomal site, corresponding to the secondary constriction. In addition, 45S rDNA, as well as telomere-associated sequences, results to be 5-methylcytosine-enriched. The results are discussed and compared with those previously obtained in other plant systems 2n = 4 with the aim to enable a better knowledge of the lengthwise differentiation of this chromosome complement.

The rat glucocorticoid receptor integration in Nicotiana langsdorffii genome affects plant responses to abiotic stresses and to arbuscular mycorrhizal symbiosis

The present study reports evidence of the pleiotropic effects caused by the insertion of the rat glucocorticoid receptor (GR) into the genome of Nicotiana langsdorffii. Transgenic N. langsdorffii-GR plants and the wild-type genotypes were analysed for their phenotypic and physiological characteristics. The integration of the GR gene affected flowering, growth habit, leaf morphology and stomatal pattern. Furthermore, GR plants showed an increased tolerance to heavy metal, drought and heat stress as evidenced by electrolyte leakage and by cell dedifferentiation and differentiation capability after recovery from stress treatments. We also monitored the establishment of the beneficial symbiosis between transgenic plants and the mycorrhizal fungus Funneliformis mosseae whose pre-symbiotic growth was significantly reduced by root exudates of N. langsdorffii-GR plants. The observed pleiotropic responses of transgenic plants may be a consequence of the hormonal imbalance, putatively due to the interaction of the GR receptor with the host genetic background. Our findings suggest that N. langsdorffii-GR plants can be used as a functional model system for the study of plant responses to a series of environmental stimuli.

DNA methylation pattern in a barley reconstructed karyotype with deleted ribosomal gene cluster of chromosome 6H

A reconstructed barley karyotype (T-35) was utilised to study the influence of chromosomal rearrangements on the DNA methylation pattern at chromosome level. Data obtained were also compared with the distribution of Giemsa N-bands and high gene density regions along the individual chromosomes that have been previously described. In comparison to the control karyotype (T-1586), the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complement. Significant remodelling process of methylation pattern was found also in the residual nucleolus organiser regions (NOR) on chromosome 5H as a consequence of deletion comprising the whole NOR of chromosome 6H in T-35. Moreover, differences between corresponding segments of the homologues with respect to some other chromosome locations were also observed. Repositioning of genomic DNA methylation along the metaphase chromosomes following chromosomal reconstruction in barley seems to be essential to ensure correct chromatin organisation and function.

Cytological characterization of Vicia oroboides Wulfen in Jacq

Vicia oroboides, a rare taxon belonging to section Atossa of subgenus Vicia, was recovered and analysed by means of cytological and karyological methods with the aim of both characterising this species and integrating our knowledge on phylogeny of subgenus Vicia. Automated karyotype analysis and nuclear DNA content have been determined after Feulgen’s reaction; chromosome banding was performed by fluorochrome staining to evidence heterochromatic blocks along the chromosome complement. The chromosome number is in line with the values of the species of section Atossa; the GC- and AT-rich sites were identified by CMA and DAPI staining. Karyomorphological parameters, based on symmetry indices, provide information about the phylogenetic position of this species inside the subgenus Vicia. DNA content is reported for the first time.

Female gametophyte and embryo development in Helleborus bocconei Ten. (Ranunculaceae).

In this study, we investigated cytohistochemistry, cycle progression, and relative DNA content of the female gametophyte cells of Helleborus bocconei Ten. before and after fertilization process. The early stages of embryo development were also investigated. H. bocconei possesses a monosporic seven-celled/eight-nucleate Polygonum type female gametophyte, characterized by a morpho-functional polarity. The cells of the embryo sac showed abundant reserves of polysaccharides, strongly increasing in the egg cell just before fertilization. With different timing in DNA replication during cell cycle progression, synergids, egg cells, and polar nuclei showed a haploid DNA content at the end of their differentiation, while antipodes underwent three DNA endoreduplication cycles. Programmed cell death symptoms were detectable in synergid and antipodal cells. After double fertilization, the central cell quickly underwent many mitotic cycles forming the endosperm, which exhibited a progressive increase in protein bodies and starch grains. Close to the developing embryo, the endosperm differentiated a well-defined region rich in a fibrillar carbohydrate matrix. The zygote, that does not start immediately to divide after double fertilization, developed in to an embryo that reached the heart stage at fruit maturation time. A weakly differentiated embryo at this time indicates a morpho-physiological dormancy of seeds, as a survival strategy imposed by the life cycle of this plant with seed dispersal in spring and their germination in the following winter.