Analisi funzionale del gene UVR8 e suo ruolo nella risposta delle piante a stress ambientali

Abstract

Plants are sessile organisms and, therefore, are continuously subjected to environmental sub-optimal or stressful conditions. In an arid environment plants are challenging multiple stresses, such, as water shortage, excessive soil salinity, osmotic stress conditions and high light intensity, including an excess of ultraviolet light mainly (UV-B). To overcome these unfavorable conditions, plants have evolved different strategies to adapt to common osmotic stress and high UV-B light. Recently, the UV-B photoreceptor, UVR8 (UV RESISTANCE LOCUS 8), has been identified and its role in the plant response to UV-B largely clarified. Besides its role in UV-B signaling, we have demonstrated that the expression of UVR8 gene is strongly induced by osmotic and salt stress in wild type A. thaliana seedling (Fasano et al., 2014). Moreover, by using a "gain and loss of function" approach we have evidenced a role of the UVR8 gene in plant growth, development and differentiation: UVR8 overexpressing plants have a reduced vegetative growth (minor diameter of the rosette, smaller leaves, height less), while silenced plants are characterized by a higher growth and produce a large number of siliques and seeds (Fasano et al., 2009; 2010), reminiscent of the response SIMR (Stress Induced Morphogenic Response). The UVR8 protein is predominantly localized in the cytoplasm and in response to low UV-B doses only a small fraction monomerizes and translocates to the nucleus, where it acts as a transcriptional activator. Most of the UVR8 protein remains in the cytoplasmic proteins and it might exert additional cellular functions by interacting with other proteins involved in the complex plant response to environmental stresses. This project was aimed at the identification of putative proteins that interact with UVR8 protein, and to establish a functional role of these interactions in plant responses to osmotic stress. The main results are summarized below: 1. by using complementary approaches of proteomics and immunoprecipitation, several potential proteins that interact with the UVR8 protein were identified; in particular, our attention was focused on the proteins APX1 (Ascorbate peroxidase) and GGT1 (glutamate-glyoxylate-aminotransferase), known for their role in the mechanisms of detoxification of H2O2, a reactive oxygen species that accumulates in the plant cell in response to different environmental conditions that generate an oxidative stress; 2. the interaction between APX1-UVR8 and UVR8-GGT1 were confirmed in vivo, by using two different assays: the BiFC and the co-immunoprecipitation; 3. through a functional analysis, it was shown that different levels of the UVR8 protein are associated with a different level of ROS, in response to conditions of osmotic stress, suggesting a possible function associated to the interaction of these between UVR8 e APX1 4. a gene expression analysis of the stress marker gene RD29 and the gene GGT1 in UVR8- knock-out or overexpressing plants was performed, in response to salt stress. These experiments provided an early indication of the effect of different levels of the UVR8 protein on the transcriptional level of these two genes and, more generally, in the global response to salt stress in Arabidopsis plants.

Further analyses are required to establish whether the interaction of UVR8 with APX1 or GGT1 might somehow influence their enzymatic activity. In addition, previous studies have shown that UVR8 binds to COP1 (an E3-ubiquitin ligase) and targets negative regulators of the UV-B dependent pathway to proteasome degradation (Huang X et al., 2013). The use of inhibitors of this proteoliytic pathway may contribute to determine whether UVR8 protein can recruit APX or GGT1 proteins in order to stabilize them or target them to the proteolytic degradation in response to direct or osmotic stress derived oxidative stress.