Regolazione trascrizionale del metabolismo della prolina in riso ( Oryza sativa L.)

As the world population grows constantly the food demand gets considerably higher. Along with the global climate changes this menace threatens food security worldwide, with a particular emphasis on developing countries. Moreover, within the scientific community there is a strong consensus that climate change–driven water scarcity, rising global temperatures and extreme weather will have severe long-term effects on crop yields. Upon exposure to water-related stresses, plants activate a wide range of molecular and cellular responses, including morphological and developmental changes, ion adjustment as well as specific transcriptional reprogramming and signal transduction. A common response triggered by low water potential and high salinity is the cellular accumulation of non-toxic compounds called compatible osmolytes (CO). CO are lowmolecular-weight metabolites, which include sugars, quaternary ammonium compounds and amino acids. Besides their role as osmoprotectant, CO have different functions such as stabilization of sub-cellular structures and free radicals scavenging. The importance of each osmolyte varies among species and following different environmental conditions, yet widely found is the imino acid proline. Proline has long been known to play a key role as compatible osmolyte following drought and salinity stress, but increasing evidence also supports its involvement in plant responses to pathogen attacks, low temperature, heavy metals, nutrient deficit as well as in the regulation of the cellular redox-state. This requires that proline accumulation is regulated by numerous and different pathways. In plants, proline can be synthesized by two routes. Under high nitrogen availability and normo-osmotic conditions, ornithine is converted into pyrroline-5-carboxylate (P5C) by an ornithine-δ- aminotransferase. On the other hand, under hyperosmotic condition proline is synthesized from glutamate, which is reduced to proline by two enzymes, namely P5C synthetase (P5CS) and P5C reductase (P5CR). The catabolism proceeds through two oxidations catalyzed by proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH), leading to the re-conversion of proline to glutamate. The transcriptional and post-translational regulation underlying this metabolism is not yet fully understood. The present work focuses on the understanding of the transcriptional regulation of proline metabolism (a particular emphasis was placed towards the biosynthetic route) and on the biochemical characterization of two enzymes involved in proline biosynthesis such as the P5CS1 and P5CS2 in rice. Gene expression is regulated by Transcription Factors (TFs) binding to different -cis regulatory elements (CRE) in the promoter region, therefore modulating transcription initiation. In order to identify different CREs in proline biosynthetic genes in rice, 1000 bp upstream of translation start site (ATG) of each gene were selected and analyzed “in silico” by using PlantPAN 2.0, a specific software for rice sequences. A total of 28 different classes of CRE were detected in OsP5CS1, OsP5CS2 and OsP5CR promoters. These elements were then used to draw a structural map as well as two theoretical models for the transcriptional regulation mediated by ABA-dependent and ABA-independent pathways. Furthermore the “in silico” analysis revealed that other hormones might act on proline biosynthesis, such as auxin. qPCR analysis was then performed to evaluate the genes expression profiles following different treatments with osmotic stress or phytohormones. Due to problems with protein purification and the low enzymatic activity observed for the P5CS enzymes, only a partial characterization has been reached. Overall, the present work contributes to shed light on the complex transcriptional regulation underlying proline biosynthesis in rice.