Dipartimento di Biologia, Ecologia e Scienze della Terra - Tesi di dottorato
Permanent URI for this collectionhttps://lisa.unical.it/handle/10955/34
Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento Dipartimento di Biologia, Ecologia e Scienze della Terra dell'Università della Calabria.
Browse
2 results
Search Results
Item Identification of epigenetic mechanisms involved in seed coat development(Università della Calabria, 2024-04-29) Talarico, Emanuela; Angelone, Tommaso; Bruno, LeonardoThe evolution of seeds is a fascinating aspect of evolutionary history and plant biology. Seeds have evolved over millions of years and are considered a significant adaptation that has contributed to the success of land plants. Seeds provide protection and a means of dispersal, enabling plants to reproduce successfully in a wide range of conditions. This evolutionary history has led to the rich diversity of plant species we see on Earth today. Seeds show remarkable adaptations to survive long journeys, including different shapes, sizes and mechanisms for dispersal. But none of these features would have been possible without the evolution of the ovule, within which sexual reproduction occurs. Indeed, ovule is the structure in which take place the formation of female gametophyte, fertilisation, embryogenesis and seed development upon fertilisation. In this scenario, the aim of this Ph. D. project was to identify, at evolutionary level, the molecular and epigenetic mechanisms involved in the ovule-to-seed switch in Ginkgo biloba plants. In particular, the focus was only on the pollination event, which in such a system is separated from fertilisation by a long time interval (i.e. four/five months). Indeed, Ginkgo biloba, a member of the gymnosperms and the only extant species of the order Ginkgoales, was used as experimental model because it is considered a living fossil due to its very ancient origins, dating back to the Permian period, the last period of the Palaeozoic, when an integument developed for the first time to cover the megasporangium. An interesting characteristic of Ginkgo, which makes it suitable for this purpose, is the production of fleshy fruit-like structures that are attractive to animals. Indeed, already after pollination, the integument takes on a consistency similar to that of mesocarp of fleshy fruit, leading to the hypothesis that it may represent a precursor to the fruit, although it cannot be identified as such because gymnosperms lack an ovary. Various approaches, including RNA sequencing, in situ gene expression, hormones localization and chromatin immunoprecipitation following by sequencing (ChIP-seq), on ovules at different stages were performed in order to identify the key pathways and the epigenetically regulated genes involved in ovule-seed switch. In order to identify the main pathways modulated by the crucial pollination event, three developmental stages of the Ginkgo ovule, collected immediately after the time frame in which pollination drop emission occurs, were used. In this context, pollination drop emission is an interesting aspect because, in many gymnosperms, it identifies the time point of possible pollen reception. Therefore, samples were collected from two different experimental fields, the first characterised by the presence of both male and female plants, and the second where only female plants are present. The two experimental fields are geographically distant from each other, which means that the plants in the second field are unable to receive pollen, so they are useful for understanding how ovule development proceeds in the absence of the pollination event. Moreover, this Ph. D. thesis was part of a larger project, which involved collaboration with the research groups coordinated by Professor Barbara Baldan, University of Padua, and Professor Lucia Colombo, University of Milan, helping to produce a large amount of data on Ginkgo, but also on Arabidopsis, which has always been the model species in plant biology. With the contribution also of the results we produced, it was possible to compare the two species and describe some of the key genes involved in ovule development in Ginkgo. Finally, most of the bioinformatic analyses related to the ChIP-seq experiment reported in this thesis were performed in collaboration with Professor Ernesto Picardi of the University of Bari Aldo Moro and Dr. Antonella Muto, post-doc in my research group at the University of Calabria.Item Impact of DNA methylation on plant growth and development: a study on a methylation-defective mutant of Arabidopsis thaliana(2017-06-09) Forgione, Ivano; Bruno, Leonardo; Van Lijsebettens, MiekeEpigenetic modifications of DNA contribute to chromatin remodeling process and gene expression regulation playing a relevant role on the development of eukaryotic organisms. DNA methylation is an important epigenetic mark consisting in the addition of a methyl group on cytosine bases, which is observed in most of the organisms at the different evolution levels. In plants, DNA methylation is controlled by several genetic pathways, encoding different methyltransferases which act on different sequence contexts. Targets for cytosine DNA methylation in plant genomes are CG, CHG and CHH (H is A, T, C) sequences. The plant DNMT1-homolog METHYLTRANSFERASE1 (MET1) maintains DNA methylation at CG sites, whereas the DNMT3 homolog DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1 and DRM2) are responsible for the de novo methylation in all sequence contexts. In addition, the plant-specific CHROMOMETHYLASE3 (CMT3) is responsible for DNA maintenance methylation at CHG sites, as well as at a subset of CHH sites. In plants DNA methylation is involved in diverse biological processes. Loss of methylation in the Arabidopsis thaliana mutants met1 and ddm1 (decrease in DNA methylation 1) causes several developmental abnormalities. Similarly, combined mutations in the DRMs and CMT3 genes induce pleiotropic defects in plants. Here, we used the Arabidopsis thaliana triple mutant drm1 drm2 cmt3, defective in DNA methylation to get deeper insight into the correlation between DNA methylation and plant growth. We identified novel developmental defects of the triple mutant dealing with the agravitropic response of the root and an altered differentiation pattern of the leaf which also exhibits a curly shape. Confocal microscopy of mutant transgenic lines expressing DR5:GFP reporter gene allowed us to verify that the loss of DNA methylation impacts on the accumulation and distribution of auxin from embryo to adult plant. The expression of auxin-related genes has been also found to be altered in drm1 drm2 cmt3 mutant. Furthermore, through an optimized and implemented protocol of comparative analysis of genomic methylated regions based on MeDIP-qPCR, we provide evidence about the direct and organ-specific modulation of auxin-related genes through DNA methylation process. The epigenetic mechanisms interplay with each other rather than work independently to modulate gene function. Accordingly, in our study we provide a novel evidence of the crosstalk between DNA methylation status and histone modification. Indeed, in the drm1 drm2 cmt3 mutant the overexpression of CLF gene, a component of PCR2 complex that performs trimethylation of histone H3 lysine 27, was accompanied by a high level of histone methylation, as evaluated through ChIP-qPCR analysis, and by a concomitant down-regulation of genes target of PRC2 complex action. Thus, the results obtained in these three years of PhD course are encouraging and may open new perspectives in the study of the DNA methylation in plants.