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
