Molecular and functional analysis of developmental genes in Arabidopsis thaliana

Doctoral Dissertation uoadl:1309203 700 Read counter

Unit:
Τομέας Βοτανικής
Library of the School of Science
Deposit date:
2016-10-03
Year:
2016
Author:
Μπερή Δέσποινα
Dissertation committee:
Κοσμάς Χαραλαμπίδης, Επικ. Καθηγητής
Original Title:
Μοριακή και λειτουργική ανάλυση αναπτυξιακών γονιδίων στο Αrabidopsis thaliana
Languages:
Greek
Translated title:
Molecular and functional analysis of developmental genes in Arabidopsis thaliana
Summary:
The WD40 motif, or beta-transducin repeat (WDR), is considered one of the most
abundant domains among the eukaryotic proteins, while it is rarely found in
bacteria. The ability of WD40 proteins to fold into a tertiaryβ-propeller
architecture has made them excellent scaffolds for protein-protein and
protein-DNA interactions. The diversity within the WD40 sequence of WDR
proteins enables them to assemble a vast number of distinct multi-protein
complexes. Due to the low conservation rate of WDR repeats, the identification
and characterization of WD40 proteins is an extremely difficult procedure.
The aim of this PhD thesis was to characterize AtULCS1(Ubiquitin Ligase
Complex Subunit 1) gene of Arabidopsis thaliana that encodes for a novel WD40
protein. AtULCS1 consists of 331 amino acids and contains five WD40 repeats
that form the structural WD40 region of the protein. In a massive in silico
analysis in Arabidopsis proteome, AtULCS1 was annotated as a putative subunit
of an E3 Cullin-Ring Ligase (CRL) complex.
During the phylogenetic analysis of the protein, we identified two homologous
amino-acid sequences in all plant species checked that are organized in two
distinct clades of the phylogenetic tree. Arabidopsis proteome contains two
homologous proteins, AtULCS1 and AtAPRF1, which share a high percentage of
identity in their amino acid sequence. However, our data (PhD thesis and
unpublished data) supports the involvement of the two proteins in distinct
complexes. AtULCS1 interacts in planta with DDB1a, the protein subunit of DWD
E3 CRL complexes, and regulates a variety of cellular and developmental
processes through the proteolytic machinery. On the other hand, AtAPRF1 is
suggested to control flowering time in Arabidopsis possibly through methylation
of FLC genetic locus (Kapolas et al., 2016).
To obtain insights into the tissue specific expression pattern, Arabidopsis
transgenic plants harboring a promoter-GUS transcriptional fusion construct,
were analyzed. The aforementioned analysis showed the activity of AtULCS1
during a variety of developmental stages and its maximum expression in flower
tissues. The localization of AtULCS1 in both nucleus and cytoplasm in
Nicotianabenthamianaepidermic cells implies the assembly of both nuclear and
cytoplasmic E3 CRL complexes.
An RNAi mediated silencing and overexpression approach was used here to
functionally characterize the AtULCS1 gene. Detailed phenotypic analysis of the
ulcs1i transgenic lines revealed the involvement of the protein in a variety of
developmental processes such as seed size regulation, cell proliferation and
differentiation, plant growth and secondary wall modification. The partial
inability of ulcs1i mutants to deposit cellulose and lignin in anther
endothecium cells leads to a sterile phenotype due to anther indehiscence.
Moreover ulcs1i plants exhibit lower secondary thickening levels in the
vascular tissue of the shoot, suggesting a functional role of the gene during
the secondary wall modification pattern of the plants. Concerning the molecular
mode of action of AtULCS1 during cell thickening, our data indicate that it
possibly regulates the expression of several MYB transcription factors that act
as “master switches” during this process. This, results into the disturbance of
the expression levels of several IRX biosynthetic genes and finally to lower
secondary wall modification of endothecium and vascular xylem cells.
Keywords:
WD40 domain, E3 Cullin-RING Ligases , Proteolysis, Cell wall, Anther dehiscence
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
130
Number of pages:
178
File:
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