A screen for new players in UVR8 signalling by reverse genetics

Diplôme Master A screen for new players in UVR8 signalling by reverse genetics eng 2022-02-09 2022-02-24 author Murphy Scott 840572 director Ulm Roman 0000-0001-8014-7392 732804 Plants are sessile organisms and must therefore constantly adapt to the biotic and abiotic conditions that surrounds them. Environmental pressures and natural selection have led to the development of different mechanisms that allow plants to detect and respond to the environment. Light plays an essential role in the development of plants and responses to the various cues it conveys are tightly regulated. UV-B is one of the wavelength ranges perceived by plants that affects its development at multiple stages. Exposure to UV-B leads to changes in photomorphogenic response such as flowering time, phototropism, root, and leaf development, as well as stomatal aperture. UV-B is a highly energetic wavelength present in solar spectrum (between 280 and 315 nm), perceived in plants by a photoreceptor known as UV RESISTANCE LOCUS 8 (UVR8). At the molecular level, UVR8 is present as a homodimer in absence of UV-B and monomerises in the presence of UV-B. Monomerised UVR8 are capable of interacting with another key element of the UVR8 signalling pathway, the CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) E3 ubiquitin ligase. COP1 in cooperation with the SUPPRESSOR OF phyA 105 (SPA) targets different proteins for ubiquitination for 26S proteasomal degradation. The UVR8-COP1 interaction inhibits the E3 ubiquitin ligase activity of the COP1-SPA complex. The ELONGATED HYPOCOTYL 5 (HY5), a target of COP1, is thus stabilised and can perform its role as a transcription factor. HY5 binds to the promoters of its target genes and initiates the UVR8-dependent response through modulation of the transcriptome. REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 (RUP1 and RUP2) are two genes regulated by HY5. RUP1 and RUP2 act in a negative feedback loop that promotes redimerization of the UVR8 photoreceptor. This study focuses on the identification and/or characterisation of new players in the UVR8 pathway. Firstly, I studied the rup2-7 mutant, expressing a novel allele of the RUP2 gene. This mutant could work as a potential tool to further understanding the multiples roles of RUP2 in the UVR8 photocycle. Secondly, a reverse genetic approach was undertaken based on a previous transcriptomic analysis, and different mutants were characterized phenotypically and molecularly in response to UV-B treatments. Anthocyanin measurements, hypocotyl length assay and Fv/Fm assay were performed to identify the responses to UV-B for 45 T-DNA insertion lines corresponding to 31 different genes. Seven genes have been identified to be potentially involved in at least one UVR8 mediated response: MAPKKK13, MAPKKK14, an Unknown protein of unknown function (UPUF), ABCI20, ELF4, MYB73 and MYB44. Finally, in light of recent publications suggesting direct UVR8 interaction with transcription factors, yeast two-hybrid experiments were performed to confirm the interaction between UVR8 and the following transcription factors: MYB73, MYB44 and WRKY36. Dewey 580 45 Département des sciences végétales 163 false 832 Groupe Ulm 554cab9e-9787-4733-ac8b-7b559ff371bb Master thesis Restricted TM_Scott_Murphy_Final.pdf 1754653 application/pdf restrictedAccess Faculté des sciences / Section de biologie / Département des sciences végétales

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A screen for new players in UVR8 signalling by reverse genetics				2022	86	7

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