Browsing by Author "Bitonti, Maria Beatrice"

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    Identification of a feral olive dehydrin gene and its development as a tool for drought tolerance in Arabidopsis thaliana
    (2013-11-28) Muto, Antonella; Bitonti, Maria Beatrice; Chiappetta, Adriana; Van Lijsebettens, Mieke
    Stress abiotici, quali deficit idrico e salinizzazione del suolo influenzano negativamente la crescita delle piante e la produttività delle colture (Liu et al., 2004; Wu et al., 2007). In campo vegetale, tra le strategie sperimentali messe in atto per incrementare la tolleranza a varie tipologie di stress tra cui siccità, salinità e congelamento, l’approccio più efficace è risultato essere l’introduzione, in piante di interesse, di geni codificanti per fattori di trascrizione stress-inducibili o, più in generale, di geni corrrelati alla risposta agli stress, di genotipi vegetali naturalmente stress-tolleranti (Beck et al, 2007). In tale contesto, Olea europaea L. subsp. europaea var. sylvestris, nota comunemente come oleastro, una pianta tipica ed ampiamente diffusa nel bacino del Mediterraneo, presenta molti tratti quali la resistenza al vento ed alla siccità, la capacità di recuperare dopo un incendio, che da una parte potrebbero essere traslati a specie vegetali di importanza agronoma ed economica rilevante, dall’altra ne fanno un candidato eccellente per le pratiche di rimboschimento e della gestione delle zone erose della Macchia Mediterranea (Mulas et Deidda, 1998) . Tra i meccanismi messi in atto dalle piante per fronteggiare stress vari tra cui quello idrico ed osmotico rientra la sintesi di una classe di proteine note come deidrine. Un membro della famiglia genica delle deidrine, denominato OesDHN è stato precedentemente identificato da una libreria a cDNA ottenuta da foglie di piante di Olea europaea subsp. europeae var. sylvestris ed interessantemente i suoi livelli di espressione sono risultati essere up-regolati in piante di oleastro esposte a condizioni di stress idrico e da freddo (Bruno et al., 2010). Le analisi volte a definirne l’omologia di sequenza e l’origine filogenetica hanno dimostrato che OesDHN codifica per una deidrina acida (pI 5.14) costituita da 211 aminoacidi di 23,846 kDa. OesDHN presenta due segmenti K ricchi in lisina ed un segmento S, ricco in serina, caratteristiche tipiche di una deidrina di tipo SK2. Inoltre, l’analisi Southern blot, condotta al fine di analizzare l’organizzazione genomica, ha dimostra che OesDHN è presente in duplice copia nel genoma aploide di oleastro. Al fine di chiarire il ruolo di OesDHN nei meccanismi messi in atto dalle piante in risposta allo stress idrico, abbiamo generato piante transgeniche di Arabidopsis thaliana overesprimenti il gene OesDHN. I risultati ottenuti hanno messo in evidenza che, in condizioni di stress osmotico medio, indotto sperimentalmente aggiungendo una concentrazione 25mM di mannitolo nel mezzo di coltura, l’overespressione del gene eterologo, incrementa la tolleranza delle piante a questa specifica condizione di stress. A conferma di tali risultati, l’analisi in silico condotta ha messo in evidenza la presenza di putativi elementi regolatori stress-inducibili di tipo ABRE e MYB, localizzati nella regione del promotore di OesDHN. Infine, l’analisi confocale sulle linee transgeniche 35S::OesDHN:GFP e 35S::GFP:OesDHN di Arabidopsis thaliana, ha messo in evidenza che la proteina OesDHN è localizzata principalmente a livello nucleare. Nel loro insieme i risultati ottenuti sulla pianta modello Arabidopsis thaliana hanno permesso di chiarire alcuni degli aspetti molecolari chiamati in causa nella tolleranza a svariate condizioni di stress, nelle piante. La prospettiva a lunga scadenza della ricerca affrontata è quella di ampliare le conoscenze utili a definire possibili strategie per incrementare caratteri di tolleranza/resistenza in importanti specie coltivate e non.
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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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    Response to light conditions in Posidonia oceanica (L) Delile plants: some cytophysiological and molecular aspects
    (2014-03-18) Bruno, Alessandro; Bitonti, Maria Beatrice; Innocenti, Anna Maria
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    <> elongator complex in plant: a study of its molecular networks
    (2014-11-29) Gagliardi, Olimpia; Canonaco, Marcello; Bitonti, Maria Beatrice; Van Lijsebettens, Mieke
    The Elongator complex is a histone acetyltransferase complex associated with RNAPII to facilitate transcript elongation. It’s composed of six proteins (ELP1-6). ELP1-3 form the Elongator core subcomplex, while ELP4-6 form the accessory subcomplex. Elongator complex, firstly identified in yeast, was later isolated from animals and plants and all its six subunits are evolutionarily conserved. The Elongator activity is conferred by ELP3 that targets specifically histons H3 (lysine-14) and H4 (lysine-12) by acetylating histone in order to facilitate the RNAPII progresses through the nucleosome. In yeast, mutations in Elongator subunits induce delay in growth due to a slowly adaptation to changing environmental conditions. In human, mutations in Elongator components affect neuronal development and this leads to neuronal disease. Whereas, in plant Elongator stimulates plant growth acting a positive regulator of cell proliferation. At the phenotypic level, Elongator mutants, called elongata, are known for narrow leaves and short root. In the present work, by using the model plant Arabidopsis thaliana, we investigated some aspects of molecular networks underlying Elongator activity and its interaction with environmental factors, mainly focusing on light conditions. Based on previous unpublished data obtained through TAP analysis, in the first period of PhD project we focused the attention on the functional study of Sec31 gene encoding a protein involved in cell secretory pathway, identified as a putative direct interactor of Elongator complex. To add information on this interaction we analyzed phenotypic and developmental characteristics of sec31 mutants to compare with elo3-6 mutant. The histological expression pattern of Sec31 and ELO3 transcripts in wild type seedlings was also investigated, through multiprobe in situ hybridization, to compare organ/tissue specific expression domains. The obtained results showed that expression pattern of the two genes is quite similar while sec31 mutants do not resemble elo3-6 phenotypes. Moreover further TAP experiments and in silico analysis of protein/protein interaction did not confirm previous data, thus excluding a direct interaction between ELO3 and Sec31. However, expression analysis in sec31 mutants of some Elongator-related genes, performed by qRT-PCR, showed that Sec31 and ELO3 share common downstream target genes and both seem play a role in auxin pathway. Future trascriptomic analyses on auxin mutants on one side, and the identification of possible interactors/players of both genes on the other side, could be useful to deepen if the molecular circuits, by which Elongator complex and the secretory machinery act on auxin pathway, show some cross-talk or they work in an independent manner. A further aspect of Elongantor molecular network that we investigated deals with role of Elongator in the skoto/photomorphogenesis pathways. In particular we investigated the elo3-6 mutant in darkness and under light condition (red, far-red and blue light) through microarray and RNA-seq approaches. Gene ontology categories over representative in elo3-6 seedlins, identified by BINGO analysis, allowed us to discover the putative targets of Elongator both in darkness and in light, and to understand the position of whole Elongator complex along either pathways. The results suggested that Elongator complex takes part in the skotomorphogenesis and photomorphogenesis and is dependent on photoreceptors PHYA and PHYB. Microarray, RNA-seq, qRT-PCR and ChIP- qPCR analyses displayed that Elongator regulates transcription of some genes both in light and in darkness. In the specific, results displayed that Elongator complex participates in the skoto/photomorphogenic pathways by binding target genes such as HYH and LHY in light and darkness condition, respectively. Whereas it can regulate the activity of other putative targets such as Pifs gene (PIF4) in darkness and HY5 under light condition.