The roles of C-TERMINALLY ENCODED PEPTIDES in Arabidopsis root development
Date
2015
Authors
Delay, Christina
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Abstract
Plants must integrate a complex array of internal and external
signals to optimise their growth in changing environments. The
ability to modulate developmental responses to varying conditions
allows them to rapidly adapt to short and long term changes in
nutrient availability. In particular, the root system shows
remarkable plasticity and can change its overall architecture to
maximise nutrient uptake. Understanding how this plasticity is
regulated could enable development of crops that are better
suited to their environment and help mitigate problems associated
with over-use of fertilisers. However, our current understanding
of how environmental information is integrated into developmental
programs is limited.
Small regulatory peptides have arisen as important regulators of
plant growth and development. They are generally thought to be
secreted into the apoplast where they can interact with receptors
on cell surfaces, thereby acting as non-cell autonomous signals.
The C-TERMINALLY ENCODED PEPTIDE (CEP) gene family, which encodes
small regulatory peptides, has 15 members in Arabidopsis. This
family is characterised by a conserved 15 amino acid peptide CEP
domain containing several residues which may be
post-translationally modified. In this thesis, the roles of the
CEP peptide family in regulating root development in response to
environmental stimuli were examined.
To initiate the study of CEPs in Arabidopsis, the relationships
between the 15 genes, their protein and peptide products and
their expression patterns were explored using in silico tools.
This formed the basis for experiments examining the expression of
CEP genes. It was found that CEPs are induced by abiotic stress
conditions, particularly nitrogen limitation, and their spatial
expression is tightly regulated. To gain an understanding of the
developmental pathways that were affected by CEP mis-expression,
transgenic plants over-expressing CEP genes were examined. It was
found that CEPs play a significant role in determining root
system architecture and also affect shoot morphology. Synthetic
peptide assays were used to corroborate these results and to
further examine the importance of the amino acid sequence and
post-translational modifications of the peptide ligand. After
confirming that CEPs are negative regulators of primary and
lateral root development, CEP3 was chosen for further in-depth
analyses.
A cep3 T-DNA insertion mutant was isolated and characterised. It
was found that this mutant was more resistant to a range of
abiotic stresses, including nitrogen limitation. The roles of
CEP3 in lateral root and primary root development were then
examined. The effect of CEP3 peptide on lateral root prebranch
site formation, founder cell specification and lateral root
primordia development was examined. This revealed that CEP3
reduces lateral root number, probably at the initiation stage. In
the primary root, excess CEP3 caused a slowing of root growth and
smaller root meristem. Reporter construct analysis revealed this
was due to a perturbation in cell cycle progression. Flow
cytometry was used to show that CEP3 peptide reduced the number
of root tip cells in the S phase of the cell cycle, whereas in
the cep3 mutant, more cells were in this phase.
Transcriptomic analysis was then used to explore the role of CEP3
in altering growth. The results indicated that CEP3 affects the
expression of genes involved in nitrogen uptake, transport and
assimilation, possibly controlling the stress induced nitrate
allocation to roots (SINAR) response. A model for CEP3 activity
was then proposed where CEP3 is induced in the roots by low N and
is perceived by one or more receptors, presumably triggering a
signalling cascade that ends in activation or repression of
specific N uptake, assimilation and allocation genes. Due to the
changes in expression of these genes, there may be a decrease in
nitrate (and nitrogen assimilate) in the roots, resulting in
decreased supply of resources to the RAM where cell cycle
progression is consequently affected. The alteration in cell
cycle results in drastically slowed root growth. This model
represents the first description of the mode-of-action of any CEP
family member in Arabidopsis.
The discovery of the role of CEP3 as mediator of root growth and
nutrient allocation has profound implications for our
understanding of plant development and nutrient use. If these
findings translate to crop species, perturbing CEP3 levels could
present an exciting avenue for controlling nutrient-distribution
and -use efficiency, reducing the over-reliance on
environmentally taxing fertilisers.
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Arabidopsis, root development, peptide, hormone, regulatory peptide, environmental cues, abiotic stress
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