Abstract |
Chapter I: Plant signaling responses can be triggered by diverse forms of abiotic stress, which are
increasingly escalated by climate change. Therefore, deciphering how plants perceive and react
to stress is becoming a highly necessary field of study. NtRBP1, a previously suggested to phaseseparate in heat stress glycine-rich protein, provides a possible link between stress response and
phase separation. The aim of my thesis was to investigate the properties of RBP1 that drive phase
separation in stress response, employing (i) an in silico approach to assess its phase separation
propensity and to identify possible post-translational modifications or disorder-to-order
substitutions that disturb biomolecular condensation, (ii) a site-directed mutagenesis approach
for the in vitro characterization of these predictions and (iii) a cell biology approach to uncover
how phase separation is functionally linked to stress response and how it affects subcellular
localization. My results showed that the predicted SUMOylation-deficient mutant RBP1K55R did
not show strikingly different subcellular localization patterns during heat and cold stress, nor it
affected alternative splicing, but was successfully purified with RBP1 for further in vitro phase
separation experiments. Additionally, in silico disorder prediction tools highlighted its phaseseparation propensity and similarity to prion-like examples of amino acid composition. Finally,
substitutions that alter predicted disorder-to-order (RBP1Y114F) transition and phosphorylation site
conservation (RBP1Y129F), showed irregular subcellular localization patterns both in room
temperature conditions and after heat stress, underlining the importance of further investigation
for their functional impact. Overall, my findings provide insights to how phase separation and
stress response are potentially linked, and highlight the importance of a more in-depth
investigation of structural and functional changes in RBP1.
Chapter II: Blue light (BL) photoreceptors are necessary components of signaling processes in plants,
regulating the growth of plants to the most optimal position for survival and growth. Modern
vertical farms utilize light-emitting diode lights with customized light to maximize efficiency,
underlining the importance of deciphering the inner workings of BL response. My study aimed to
contribute to the understanding of how Kin 7.3, a motor-based microtubule protein that was
found to associate with BL photoreceptor PHOT1, is involved in blue light (BL) induced signaling
response. Specifically, my thesis combined two approaches (i) genetic studies to explore the
impact and the mechanism of PHOT1- dependent response to BL and (ii) cell biology experiments
to study the effect of Kin7.3 on PHOT1 localization patterns and microtubule reorganization.
Results showed that loss-of-function mutants display insensitivity to BL-induced bending, which
poses a key phototropic response. Furthermore, PHOT1 distribution patterns at the plasma
membrane (PM) appeared altered by the lack of Kin7.3, with PHOT1-GFP to present increased
levels and retardation in internalization. Additionally, lack of Kin7.3 seems to affect microtubule
BL-induced organization, with microtubules showing resistance to revert from a longitudinal to
a transverse pattern of organization after BL-induced reorientation. Combined with experimental
evidence suggesting that Kin7.3 affects the phosphorylation status of PHOT1, these assays
collectively highlight Kin7.3 as a novel component of the PHOT1-dependent signaling.
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