Dipartimento di Biologia, Ecologia e Scienze della Terra - Tesi di dottorato

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Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento Dipartimento di Biologia, Ecologia e Scienze della Terra dell'Università della Calabria.

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    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, Mieke
    Epigenetic 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.
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    Impact of DNA methylation status on plant response to cd explored through a transcriptomic analysis
    (2019-04-11) Pacenza, Marianna; Bitonti, Maria Beatrice; Cerra, Maria Carmela
    Due to their sessile life style, plants are continuously exposed to a variety of abiotic and biotic stresses which could potentially hinder their growth, development, productivity and survival. In this scenario, it appears evident the relevance of epigenetic mechanisms in assuring growth plasticity to the plant and withstanding stresses through a rapid and extensive modification of gene expression in a manner that overcomes the restrictions of a highly stable DNA sequence. Epigenome landscape is largely related to DNA methylation process, which is one of the most significant players in the control of plant responses to environmental changes and stressors. On the other hand, all these responses are also under the control of an intricate signalling network which strongly involves the phytohormones, whose action is in turn influenced by epigenetic mechanisms. Despite this information, the complex mechanisms by which DNA methylation modulates plant stress responses are yet largely unresolved, mainly with respect to heavy metal stress, for which a metal- and speciesspecific response was evidenced. In order to gain further insight into these aspects, in the present work we performed a comparative transcriptomic analysis on the drm1 drm2 cmt3 (ddc) mutant of A. thaliana, defective in both maintenance and de novo DNA methylation, and WT plants exposed to a long lasting (21 days) Cd treatment at 25 and 50 μM concentrations. Attention was focused on Cd as one of the most toxic pollutants, widespread in both terrestrial and marine environment. The mutant was chosen as a suitable tool for investigating mechanisms and molecular processes that act in and are regulated by DNA methylation. Analyses of growth parameters and targeted cytophysiological features were also carried out. Concerning the results, transcriptomic analysis highlighted photosynthesis, stress responses and hormone biosynthesis as the genetic pathways more impacted by Cd treatment in both ddc mutant and WT. All these pathways are highly relevant for plant development. A more detailed analysis carried out on the pathways related to the phytohormones suggested that, under a prolonged heavy metal exposure, plant activity was directed to enhance and/or maintain the level and signalling of hormones which are relevant in sustaining the growth (auxins, cytokinins and gibberellins) more than those of hormones specifically related to stress response (jasmonic acid, abscisic acid and salicylic acid). This could represent the plant strategy to avoid the negative effects of long-lasting activity of stress-related hormones. Interestingly, such strategy could be more efficient in ddc mutant than in the WT. Indeed, likely due to a higher genome plasticity conferred to the mutant by its DNA hypomethylated status, in the ddc mutant the described transcriptomic differences have already been observed in the treatment with 25 μM Cd, while in the WT only in the treatment with 50 μM Cd. The outcome of this different modulation of gene expression was a better growth performance in ddc vs WT, as evidenced by growth parameters analysis. A tight relationship between the hormonerelated transcriptomic differences and the different cyto- morphophysiological features of ddc mutant vs WT under Cd treatment was also revealedUniversità della Calabria, Dipartimento di Biologia, Ecologia e Scienze della Terra. Dottorato di Ricerca in Scienze della Vita. Ciclo XXXI SSD BIO/01
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    DNA methylation patterns in aging and aging phenotypes
    (2019-04-11) Guarasci, Francesco; Bellizzi, Dina; Cerra, Maria Carmela
    During my PhD program, my work has been addressed to the study of the role of epigenetic modifications in aging and in age-related phenotypes. Epigenetics is the study of changes in gene expression that do not involve changes to the underlying DNA sequence. These changes affect cellular phenotypic expression by regulating relative gene expression levels. They are a common and natural process in living cells and are tightly controlled by pre-programmed mechanisms. Epigenetics modifications can be influenced by multiple factors including environmental conditions, lifestyle, nutrition, use of drugs, disease state and age. Patterns of DNA methylation, the best known and characterized epigenetic modification, change during aging; indeed, with increasing aging, genome-wide methylation levels decrease, meanwhile genomic regions, including CpG islands, become more methylated. Analyses of the above patterns provided new perspectives for establishing powerful biomarkers of human aging which have the potential to generate accurate prediction not only of the chronological but also of the biological age. The first section of the PhD thesis consists in a comprehensive overview of the general features of DNA methylation and its implication in age and age-related diseases. The topic is addressed referring to the methylation patterns established not only at nuclear but also at mitochondrial genome level. In addition, the influence of a number of environmental factors on the above patterns is also discussed. In the second section, an original research work, carried out in order to identify novel biomarkers of aging, is reported. In this work, methylation status of nuclear genes involved in mitochondrial fusion, fission, biogenesis and mitophagy, fundamental components of the mitochondrial quality control process, was investigated in subjects of different ages of the Calabrian population. The methylation levels of RAB32 and RHOT2 genes were significantly associated with age and, in particular, those of RAB32 even with the risk of developing disability. The study, therefore, led to the identification of two new biomarkers for both chronological and biological aging. In the Appendix, research works already published are reported. The first one concerns the correlation between DNA methylation and nutrition during lifetime. Global DNA methylation profiles were examined in different tissues of rats of different ages, fed with a standard and hypocaloric diet, and their association with aging and nutrition was evaluated. The results obtained have shown that tissue-specific variations in methylation levels occur during aging and that nutrition influences the state of global DNA methylation during the course of life. The hypocaloric diet seems to influence more strongly the epigenetic status of the offspring when administered during the maternal pre-gestational period compared to the gestation and lactation period. Therefore, changes in the global DNA methylation status represent an epigenetic mechanism by which age and nutrition intersect each oth and, in turn, influence the plasticity of aging. The second one is a review on the current advances in mitochondrial epigenetics studies and the increasing indication of mtDNA methylation status as an attractive biomarker for peculiar physiological and pathological phenotypes. It comes from the increasing evidence on the fact that, similarly to nuclear DNA, also mtDNA is subject to methylation and hydroxymethylation and these modifications are influenced by multiple environmental factors.
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    Heteroplasmy of the mitochondrial DNA control region in families of centenarians
    (2006-10-05) Scornaienchi, Vittorio; Rose, Giuseppina; De Benedictis, Giovanna
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    Expression profiles of stress-responder nuclear genes in relationship to common mitochondrial DNA variability
    (2013-10-28) Taverna,Daniela; Bellizzi,Dina; De Benedictis,Giovanna
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    MtDNA heteroplasmy in longevity: a puzzling story
    (2013-10-28) Romeo,Giuseppe; Rose,G.; Dato,Serena; De Benedictis,Giovanna