The roles of C-TERMINALLY ENCODED PEPTIDES in Arabidopsis root development

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. Show more