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

Mirko Pavicic

Research output: ThesisDoctoral Thesis


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 RINGtype
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.
Original languageEnglish
Awarding Institution
  • Doctoral school in environmental, biological and food sciences
  • Himanen, Kristiina, Supervisor
Award date4 Dec 2019
Place of PublicationHelsinki
Print ISBNs978-951-51-5482-8
Electronic ISBNs978-951-51-5483-5
Publication statusPublished - Oct 2019
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • 1181 Ecology, evolutionary biology

Cite this