Browsing by Author "Van Lijsebettens, Mieke"

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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.
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.
<> 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.