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Characterization of the arabidopsis thaliana myb59 transcription factor
Full text linkLe piante sono frequentemente sottoposte a stress, ossia a condizioni esterne che influiscono negativamente sulla crescita e sullo sviluppo. La percezione dello stress e la trasduzione del segnale che portano all’attivazione di risposte adattive sono passaggi cruciali nel determinare la sopravvivenza della pianta. Le piante possono rispondere ad uno stimolo esterno attivando meccanismi di difesa specifici per un determinato stress, oppure meccanismi in grado di rispondere a più stress. Infatti, le varie vie di trasduzione del segnale sono spesso interconnesse fra loro a vari livelli, possono condividere uno o più componenti o intermedi oppure avere output comuni (Chinnusamy et al., 2003). Le informazioni sul cross-talk comunque sono ancora limitate, in quanto poco è noto riguardo ai recettori e agli intermedi delle vie di trasduzione del segnale. A valle della cascata di reazioni si trovano i fattori di trascrizione che sono in grado di regolare l’espressione genica legandosi al DNA in maniera sequenza-specifica. Tali proteine sono normalmente espresse in maniera tessuto-specifica, stadio di sviluppo-specifica o in modo dipendente da uno stimolo esterno, come ad esempio uno stress ambientale, e sono responsabili della selettività nella regolazione genica (Zhang, 2003). Quindi, caratterizzare i fattori di trascrizione può essere un passo importante verso la comprensione dei meccanismi che stanno alla base dei processi di sviluppo e di risposta agli stress. La famiglia di fattori di trascrizione più rappresentata in pianta è la famiglia MYB (Stracke et al., 2001). I membri di tale famiglia sono caratterizzati dalla presenza di un dominio strutturalmente conservato di circa 52 aminoacidi (dominio MYB), contenente tre residui di triptofano spaziati regolarmente. Tali proteine sono in grado di legare il DNA in maniera sequenza specifica adottando una conformazione helix-turn-helix. I diversi membri della famiglia vengono identificati sulla base del numero di repeats imperfette di tale dominio, che può essere ripetuto fino a tre volte. Sono state proposte diverse funzioni per i diversi fattori MYB: alcuni sono coinvolti nel controllo del metabolismo secondario, della proliferazione e del differenziamento di alcuni tipi di cellule, mentre altri sono necessari nelle vie di trasduzione del segnale che rispondono a differenti stimoli (Martin e Paz-Ares, 1997). In particolare, i fattori di trascrizione MYB sembrano essere importanti nella mediazione delle risposte a molecole segnale come l’acido salicilico, e ad ormoni come l’acido abscissico e l’acido gibberellico. Lo scopo di questo progetto di dottorato è stato quello di caratterizzare il gene MYB59 di A. thaliana che appartiene alla famiglia R2R3-MYB ed è presente in tre varianti di splicing, indicate per semplicità MYB59.1, MYB59.2 e MYB59.3. È noto che il gene in esame è coinvolto nella Riassunto VI risposta allo stress da cadmio (Li et al., 2006) ed, in particolare, è stato dimostrato che l’espressione del gene omologo in B. juncea viene modulata dopo l’esposizione a tale metallo (Fusco et al., 2005). Tramite analisi Real-Time PCR, condotta su piante di Arabidopsis WT sottoposte a trattamento con Cd, è stato confermato un coinvolgimento di tale gene nella risposta a questo tipo di stress; infatti la sua espressione veniva indotta dopo 2 ore di esposizione al Cd. Per verificare se la trascrizione di questo fattore MYB subisce una modulazione anche in seguito ad altri stress di tipo abiotico, piante di A. thaliana WT sono state sottoposte a differenti trattamenti: alte e basse temperature (42 °C e 4 °C), stress idrico, stress salino (NaCl) e trattamenti ormonali (IAA, ABA, kin e GA3). L’analisi Real-Time PCR ha mostrato che la variante MYB59.2 rispondeva allo stress da ABA, freddo e disidratazione, mentre l’espressione della variante MYB59.3 veniva indotta dalla disidratazione sia nelle foglie sia nelle radici. L’espressione della forma MYB59.1, invece, non presentava modificazioni significative in seguito a tali trattamenti. In seguito, è stata condotta un’analisi dell’espressione genica, tramite Real-Time PCR, in piante di Arabidopsis WT in diversi organi: foglie della rosetta, foglie caulinari, stelo, fiori chiusi, fiori aperti ,e radici. L’analisi ha previsto l’utilizzo di tre coppie di primers, ognuna specifica per una delle tre varianti di splicing del gene stesso. L’analisi ha mostrato che le tre varianti di splicing avevano pattern di espressione organo-specifica. La caratterizzazione del gene è proseguita con l’isolamento del cDNA full length di MYB59.1, MYB59.2 e MYB59.3 ed il clonaggio nel vettore pMD1, sotto il controllo del promotore CaMV35S, per l’espressione in pianta. Inoltre, è stato analizzato un mutante inserzionale knock-out per il gene MYB59. Sono state messe a confronto piante WT, piante sovraesprimenti le tre forme di splicing e piante mutanti knock-out. Si è notato che le piante sovraesprimenti MYB59.1 presentavano un’area fogliare maggiore, mentre le piante mutanti avevano foglie con dimensioni molto minori rispetto a piante controllo. Anche l’espressione ectopica di MYB59.2 e MYB59.3 portava ad una diminuzione dell’area fogliare. Successivamente, è stato confermato il coinvolgimento di MYB59 nella risposta al Cd. È stata eseguita la quantificazione del contenuto di tale metallo in foglie e radici di piante WT, sovraesprimenti le tre forme di splicing e piante che presentavano una riduzione del 98% dell’espressione genica (dovuta all’induzione del silenziamento genico). I dati indicavano che il gene potrebbe essere coinvolto nel trasporto del Cd dalle radici alle foglie, in quanto, nelle radici, le piante con espressione ectopica del gene presentavano un contenuto del metallo maggiore rispetto a quella riscontrata nelle piante controllo. Nelle foglie invece la quantità di Cd era maggiore nelle linee silenziate. Successivamente è stato eseguito lo studio delle sequenze promotrici. Sono state quindi amplificate tre regioni di circa 2.0 Kbp a monte degli ATG delle tre forme e sono state clonate in un vettore a monte del gene reporter GUS. Il clonaggio del promotore della variante MYB59.1 ha previsto l’inserimento di due mutazioni Riassunto VII puntiformi all’interno della sequenza amplificata, in quanto l’ATG di tale forma si trova a valle rispetto agli ATG delle altre due. Attraverso un set di tre reazioni di PCR effettuate con primers contenenti le mutazioni volute, gli ATG delle varianti MYB59.2 e MYB59.3 sono stati modificati in TAG. Il saggio istochimico dell’attività GUS ha permesso di analizzare l’espressione delle tre varianti geniche nei diversi organi e tessuti vegetali. L’espressione di MYB59.1 è stata localizzata soprattutto a livello delle nervature fogliari, mentre l’espressione di MYB59.2 principalmente nelle antere immature, e per MYB59.3 si è osservata l’espressione in tutti gli stadi vegetativi e nei sepali, ma non nelle antere. Si può quindi ipotizzare che le tre varianti di splicing, avendo tre diverse localizzazioni, possano avere anche distinte funzioni all’interno della pianta. È stato inoltre analizzato il pattern di metilazione di una regione ripetuta (direct repeat) che si trova nell’intorno del TATA box e della regione che contiene l’intera sequenza del primo introne. L’analisi è stata condotta in foglie e antere di piante di Arabidopsis WT usando il metodo del bisolfito, che converte le citosine non metilate in uracile. Le foglie presentavano una metilazione maggiore rispetto a quella delle antere. Per verificare se esiste una corrispondenza tra mRNA e proteine, e quindi per capire quale variante di splicing viene in effetti espressa in pianta, sia in condizioni di crescita standard sia in seguito a stress, sono state adottate due diverse strategie, che hanno previsto la preparazione di proteine di fusione con il FLAGtag e con l’HaloTag®.Plants are frequently subjected to stress, that is external conditions that negatively influence
plant growth and development. The perception of stress and signal transduction, carrying to the
activation of adaptive responses are critical steps in determining plant survival. Plants can
respond to an external stimulus activating defence mechanisms specific for a particular stress,
or mechanisms able to respond to different stresses. In fact, different signalling transduction
pathways are often interconnected to various levels, can share one or more components or
intermediates or have common outputs (Chinnusamy et al., 2003). However, the information
about the cross-talk is limited, since little is known about signalling pathways receptors and
intermediates. Downstream of the reaction cascade there are transcription factors, that are able
to regulate gene expression binding DNA in a sequence-specific manner. These proteins are
normally expressed in a tissue, development or stimulus-specific manner, such as an
environmental stress, and are responsible for the selectivity in gene regulation (Zhang, 2003).
So, understanding TFs function is an important step towards studying plant development and
stress responses. MYB transcription factors family is the larger TFs family in plants (Stracke et
al., 2001). Its members are characterized by a structurally conserved domain of about 52
aminoacid (MYB domain), containing three regularly spaced tryptophan residues. They are able
to bind DNA in a sequence-specific manner adopting an helix-turn-helix conformation. MYB
proteins can be classified into three groups depending on the number of adjacent repeats in the
binding domain. Different functions was proposed for different MYB proteins: some are involved
in the control of secondary metabolism, cell proliferation and differentiation, whereas others are
needed in signal transduction pathways responding to different stimuli (Martin e Paz-Ares,
1997), such as salicylic acid, and abscisic and gibberellic acid.
This PhD work has been focused on the characterization of the AtMYB59 gene, that belongs to
the R2R3-MYB family and presents three splicing variants, called for simplicity MYB59.1,
MYB59.2 e MYB59.3. It is known that this gene is involved in the Cd stress response (Li et al.,
2006) and, in particular, it has been demonstrated that the expression of its gene homolog in B.
juncea was modulated after Cd exposure (Fusco et al., 2005). Through Real Time PCR, carried
out on Arabidopsis WT treated with Cd, an involvement of the gene in the response to this type of
stress was confirmed; in fact, the expression was induced after a 2 h Cd treatment. To verify if
the gene transcription was modulated also after different abiotic stress, Arabidopsis WT plants
were subjected to different treatments: high and low temperatures (42 °C and 4 °C), drought,
Summary
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salinity (NaCl) and hormonal treatments (IAA, ABA, kin e GA3). Real Time PCR analysis was
shown that MYB59.2 responded to ABA, cold and drought stress, whereas MYB59.3 was induced
by drought in leaves as well as in roots. MYB59.1 expression, instead, did not show important
modulations after these treatments. Furthermore, through Real Time PCR, gene expression
analysis on different Arabidopsis organs was conducted: rosetta leaves, cauline leaves, stem,
closed flowers, open flowers and roots. For this analysis primers designed on a unique region of
each splicing variant were used. The results indicated that the three splicing variants had
different organ specific expression. Gene characterization was carried out by isolating full-length
cDNA of MYB59.1, MYB59.2 and MYB59.3 and cloning in pMD1 vector for plant expression, under
the control of CaMV35S promoter. Moreover, an insertional knock-out mutant was analyzed.
Comparison between WT, overexpressing and mutant plants showed that plants overexpressing
MYB59.1 had a leaf area higher and mutant plants lower than control plants. The overexpression
of MYB59.2 and MYB59.3 also induced a decrease of leaf area in respect to WT plants.
Afterwards, an involvement of MYB59 in Cd response was confirmed. The quantification of Cd
content in leaves and roots of WT, overexpressing and plant showing a gene expression
reduction of 98% (due to gene silencing induction) was carried out. Data indicated that the gene
may be involved in root-to-shoot Cd transport, since, in roots, overexpressing plants showed a
metal content higher than that in control plants. In leaves, instead, Cd content was higher in
silenced lines. Subsequently, a study of promoter sequences was carried out. Three region of
about 2.0 Kbp upstream ATGs of the three forms were amplified and cloned in a vector upstream
a GUS gene reporter. For the analysis of the promoter region regulating MYB59.1, mutagenesis of
the ATG of MYB59.2 and MYB59.3, that are upstream the starting codon of this variant, was
performed. So, these two ATGs were converted in the TAG stop codons, through a set of three
PCR reactions. GUS assay allowed to localized the expression of the three variants in different
plant organs and tissues. The expression of MYB59.1 was found mainly in leaf veins; the
expression of MYB59.2 was mainly in the immature anthers, whereas the expression of MYB59.3
was localized in most vegetative tissues, sepals, but not in anthers. It can be hypothesized that
the three splicing variants, having different localizations, may also play different roles in plant.
Moreover, methylation pattern of a direct repeat around the TATA box and the region containing
the whole first intron sequence was analyzed. The analysis was carried out in leaves and anthers
of Arabidopsis WT using bisulfite method, that converts unmethylated C in U. In leaves, this
repeat region is highly methylated, whereas not in anthers.
To verify if a correspondence between mRNA and proteins exists, and so to understand which
splicing variants is actually translated in plant, in standard condition as well as after stress
exposure, two protein fusion strategies were carried out using the FLAGtag and the HaloTag®.
Summary
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Unfortunately, in both cases Western blot analysis showed aspecific signals. To avoid this
problem, the preparation of an antibody anti-MYB59 is under preparation. It has been reported
that MYB59 share the 74.2% nucleotide sequence identity in their coding region with MYB48,
another R2R3-MYB. The two genes show a conserved alternative splicing mechanism and
probably are the result of a relatively recent duplication event (Li et al., 2006). Since myb48
mutant plant did not show phenotypic differences respect to WT, single mutants knock out were
crossed to obtain double mutant myb59myb48, to better understand the role of these two TFs in
plants.
Understand MYB59 gene function may be an important step to gain insight into plant
development and stress response mechanisms
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