Biochemical characterization of the PARP domain-containing proteins AtRCD1 and TaSRO1

Abstract

Quick and finely tuned response to the different abiotic stresses are an essential feature for the survival of of plants as sessile organisms. The signalling pathways that determine tailored responses at cellular and systemic level need to be strictly regulated, as well as coordinated, in order to allow a fast rise of defensive mechanisms. A pivotal role in the integration and regulation of different molecular pathways is played by proteins defined as “cellular hubs”: an example of these proteins is A. thaliana RADICAL-INDUCED CELL DEATH1 (RCD1), the best characterized member of the plant-specific SIMILAR TO RCD1 (SRO) protein family. It was first described to be involved in apoplastic ROS tolerance and development, as well as in response to heat and chloroplastic ROS stress, via interaction with different transcription factors through its RST domain. However, the mechanism, by which the interaction between RCD1 and its targets is regulated, has not been uncovered yet. In this work, the biochemical characterization of the function of the WWE domain, located at the N-terminus of RCD1, was carried out, in order to uncover its functions in the regulation of RCD1 activity in the plant cell nucleus. The localization study performed with different RCD1 constructs showed how the WWE domain has a major influence on the characteristic subnuclear localization of RCD1 in nuclear bodies, present in different sizes and number. The Co immunoprecipitation assays confirmed the role of the N-terminus of RCD1 and its closest homologue, SIMILAR TO RCD1 1 (SRO1), including the WWE domain, in the oligomerisation process, that could be involved in the formation of the nuclear bodies. Alteration of previously identified phophosites on the N-terminal portion of RCD1 were generated to test whether the phosphorylation state of the considered phosphorylatable residues might play a role in the regulation of RCD1’s activity in the plant cell. In this work it was shown that mutations on phosphosites produced noticeable effects both on the macroscopic and on the molecular level. Additionally, in this work another member of the SRO protein family, Triticum aestivum SRO1 was studied, in relation to the previously described catalytic activity of its Poly(ADP)-ribose Polymerase (PARP) domain, that would make this the only known member of the SRO family to present PARP enzymatic activity. However, with the assays performed in this work, the previously published findings could not be confirmed.