Multifaceted roles of RING-type ubiquitin E3 ligases : reverse phenomic approaches

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Title: Multifaceted roles of RING-type ubiquitin E3 ligases : reverse phenomic approaches
Author: Pavicic, Mirko
Other contributor: Helsingin yliopisto, maatalous-metsätieteellinen tiedekunta
Helsingfors universitet, agrikultur-forstvetenskapliga fakulteten
University of Helsinki, Faculty of Agriculture and Forestry
Kasvitieteen tohtoriohjelma
Doktorandprogrammet i botanik
Doctoral Program in Plant Sciences
Publisher: Helsingin yliopisto
Date: 2019-10-02
Language: en
Belongs to series: URN:ISSN:2342-5423
Thesis level: Doctoral dissertation (article-based)
Abstract: Ubiquitin proteasome system (UPS) is an ATP dependent pathway for targeted protein degradation. The role of UPS is to maintain a healthy protein balance in the cell and to mediate activation and repression of plant developmental processes, hormones and other signalling cascades as well as responses to environmental perturbations. The UPS is composed of several actors, the most important of them being the ubiquitin E3 ligases, which are responsible for providing the specificity for substrate recognition. About 5% of Arabidopsis thaliana genome encodes for ubiquitin E3 ligase genes (~1.400), classified in seven different subgroups, among which the second most abundant group is the RING-type ubiquitin E3 ligase family with nearly 500 members. More than half of the RING-type ubiquitin E3 ligase genes are uncharacterised. Those that are characterized demonstrate their multi-target ability which implies additional roles and cross-reactivity with other pathways. With the emergence of high throughput sequencing, improved Arabidopsis genome assemblies are available and there is a constantly growing amount of transcriptomics data available for the RING-type ubiquitin E3 ligase genes that link them to different developmental stages and perturbations. However, only few of these genes have been associated phenotypically with these processes. Our first aim was to use reverse genetics approach to rescreen Arabidopsis genome in order to update the number of annotated RING-type ubiquitin E3 ligase genes. We further aimed to develop a set of image-based phenotyping methods to systematically assign them in their signalling cascades and developmental pathways, and to functionally characterize the identified molecular networks of the RING-type ubiquitin E3 ligases and their substrates. This study revealed 50 new RING-type ubiquitin E3 ligases genes, while 31 earlier annotated genes were excluded, giving a total new count of 509 RING-type ubiquitin E3 ligases genes. RING-type ubiquitin E3 ligases were then assigned to different developmental, hormonal and/or perturbation related pathways, based on their gene expression profiles. To allow systematic and efficient functional confirmation of these associations in plants, protocols for image-based high throughput phenotypic assays were established. In these assays, the associated knockout lines were studied for rosette shape and growth, cotyledon emergence as a proxy for germination analysis, and Botrytis cinerea symptom progression. These phenotypic screens confirmed 36 flower enriched RING-type ubiquitin E3 ligases, 11 of which were associated to flowering, three rosette and one sepal development. Four of them responded negatively and one positively to ABA treatment at germination. Furthermore, ten RING-type ubiquitin E3 ligases were associated with Botrytis responses, with one characterised at molecular level. Collectively, the results of this study demonstrated the versatility and pleiotropy of RING-type ubiquitin E3 ligases and set the foundation for a systematic screening of phenotypes regulated by UPS components.RING-tyyppisen ubikitiin E3 -liigaasien monipuoliset roolit: käänteiset fenomiset lähestymistavat
Subject: Plant Biotechnology
Rights: Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.

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