Identification of regulatory elements responsible for metal hyperaccumulation in the Brassicaceae family. Functional analysis of the Arabidopsis thaliana MYB48 and MYB59 transcription factors.

Chapter 1: Identification of regulatory elements responsible for metal hyperaccumulation in the Brassicaceae family. The role of the metal transporter MTP1 in metal tolerance and accumulation has been extensively studied, due also to its great importance in the hypertolerance trait. MTP1 is known to have undergone copy number expansion in hyperaccumulator Arabidopsis halleri; moreover, possible differences in cis-regulation between hyperaccumulators and non-accumulators have been proposed. This work focuses on the analysis of the MTP1 promoter. The expression pattern and levels driven by the Arabidopsis thaliana and A. halleri promoter sequences are markedly different, coherently with the different accumulation ability and metal storage tissues displayed by the two species. MTP1 expression in roots was found in both species and is associated with the presence of root-specific cis elements in both promoters. Similarly, guard cell-specific expression was observed for both A. thaliana and A. halleri sequences and is associated with the presence of Dof-binding sites. In addition, the MTP1 promoter of A. halleri drives expression in trichomes. This interesting localization is likely associated to a couple of MYB-binding sites in the 5’UTR of the gene. Metal accumulation in trichomes is an intriguing feature in A. halleri and is possibly involved in short-term tolerance to metals. Chapter 2: Functional analysis of the Arabidopsis thaliana MYB48 and MYB59 transcription factors. MYB transcription factors are involved in many events of plant life, as cell differentiation and metabolism, plant development, response to hormones and to environmental stimuli. Among the others, MYB48 and MYB59 have been proposed to participate in secondary development, cell cycle regulation and response to abiotic stress in Arabidopsis thaliana. In this work, a myb48myb59 double mutant was used due to probable functional redundancy of the two transcription factors. myb48myb59 plants show smaller rosette leaves, likely due to reduced cell distension, delayed flowering and longer roots in comparison to wt; early senescence was also considered and confirmed by the higher SAG12 expression levels. The phenotype is consistent with a reduced cytokinin content: this observation was confirmed by the increased sensitivity to exogenous cytokinins and by the modulated genes resulting from the microarray experiment.