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Georgios Kepesidis

Investigating Phototropin-dependent responses in Chlamydomonas reinhardtii

Published on 1 June 2022
Thesis presented June 01, 2022

Light is crucial for life on earth, especially for photosynthetic organisms; it is a source of spatiotemporal information perceived by photoreceptor proteins, as well as the energy source that fuels photosynthesis. However, when absorbed in excess, it can be toxic for the photosynthetic cells leading to reactive oxygen species formation; this is avoided by the photoprotective mechanism qE (quenching of energy). Chlamydomonas reinhardtii is a model unicellular green alga, which is widely used for genetic and cellular studies, including those concerning light perception and utilization. Chlamydomonas accurately senses the information provided by light and regulates important cellular functions, including gene expression, sexual life cycle, phototaxis, photosynthesis, and photoprotection, using a network of specialized photoreceptors. It is equipped with a single-copy phototropin, four cryptochromes, eight rhodopsin-like proteins, as well as the UV-B photoreceptor UVR8.
Among these photoreceptors, phototropin (PHOT) appears to have a multifaceted role in Chlamydomonas physiology; it has been shown to control gametogenesis at low nitrogen availability, expression of genes encoding chlorophyll and carotenoid biosynthesis, the size of the eyespot and photoprotection. Specifically on the later, under high light PHOT regulates positively the induction of LHCSR3, a nucleus-encoded and chloroplast-localized protein, which is the main actor of qE.
PHOT is a blue-light activated, plasma membrane associated Serine/ Threonine kinase, that undergoes phosphorylation upon irradiation with UVA and blue light. Only a handful of substrates of PHOT have been identified in higher plants, but not in Chlamydomonas. My goal was to elucidate the PHOT-dependent responses in Chlamydomonas reinhardtii and to identify possible interactors of the protein. For this, I employed a strategy involving comparative phosphoproteomic analyses, as well as targeted mutant strain screening.
On this Thesis, I am presenting data from two comparative phosphoproteomic analyses. On the first I compared the phosphoproteome of low- and high-light acclimated WT and PHOT-deficient cells (Δphot). The second phosphoproteomic analysis allowed the comparison of the phosphoproteome of dark-acclimated and shortly blue-light-exposed wild-type (WT) and Δphot cells in order to elucidate the early PHOT-dependent phosphorylating events.
I am also presenting the involvement of a PHOT-regulated serine/threonine protein kinase, hereafter FLKIN, in the control of LHCSR3 accumulation and the Carbon Concentrating Mechanism (CCM). The mutant lacking FLKIN induces the transcripts of proteins involved in the CCM in higher levels than the WT. Additionally, the Δflkin strain shows a growth defect in autotrophic medium, as well as bigger cell size and higher chlorophyl content. Δflkin has also higher photosynthetic capacity than the WT in all growth conditions.
I, finally, revealed the involvement of PHOT in the Chloroplast Unfolded Protein Response (cpUPR) in response to proteotoxic stress.

Photoperception, light signalling, photosynthesis, phototropin