Characterization of the arabidopsis thaliana myb59 transcription factor

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 IX 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 X 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.