LA ZONA EQUIPOS

Plants have clearly evolved intricate systems for integrating developmental programs with external information. The crosstalk between hormones, signalling peptides, transporters and nutrient allocation pathways is complex. At best, they are understood in the context of only one or two players. To tackle challenges presented by changes in

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climate, environmental degradation and increasing demand for food, it is imperative that these systems are better understood. In particular, the role of small signalling peptides in the developmental response to environmental information presents an exciting but under-studied avenue for RSA modulation.

Of the known small regulatory peptide families, the CEP family is the least well- studied. The founding five members of this family in Arabidopsis, discovered using an

in silico approach, were characterized by a conserved 15 amino acid peptide domain at

or near the C-terminus (Ohyama et al., 2008). The mature product was shown to be a 14 or 15 amino acid peptide containing one or two hyroxylated proline residues and the 15 amino acid peptide was reported to be biologically active on roots. Over-

expression of AtCEP1, which was mainly expressed in the shoot apical meristem and LR primordia during development, resulted in reduced primary and lateral root elongation as well as a smaller shoot system. Confocal imaging showed that CEP1 over-expression roots had a reduced number of meristem cells (Ohyama et al., 2008) .

Aside from the above study on CEP1, little was known about the CEP family. This is not suprising, as generally the characterisation of peptide families has been hampered for a myriad of reasons including i) the small size of peptide-coding genes, meaning that they are not normally picked up by annotation algorithms ii) the lack of mutants (T- DNA insertion, EMS, etc.) due to this small size, iii) some level of functional redundancy in these large multigene families and iv) lack of similarity in the precursor sequence presenting a challenge for knock down of multiple genes with one construct.

A number of tools and strategies have been used to increase our understanding of the roles of signalling peptides. For example, peptides from the CLE and RGF families have been implicated in different aspects of root development, however specific function and mechanistic action of other family members remains to be elucidated. In a step towards this, the expression patterns and effects of over-expression of all RGF and CLE peptide family members have been assessed (Jun et al., 2010; Fernandez et al., 2013). One particularly useful tool in signalling peptide research is the application of

synthetically synthesised peptides to the growth medium, similar to supplementing growth medium with synthetically derived homones. This technique was first used to elucidate the role of the CLV2 receptor in the CLE19-mediated consumption of the root

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meristem (Fiers et al., 2005). A 14 amino acid peptide was chemically synthesised and applied to growth medium at various concentrations to determine biological activity (Fiers et al., 2005). This technique has since become routinely used in the eludication of the roles of singalling peptides in plants. However, it must be noted that, similar to over-expression, appplication of synthetic peptide to the entire plant can cause off- target effects and may not necessarily provide biologically relevant insights.

Additionally, the importance of post-translational modification(s) in mediating interactions and functions has been reported and synthetic peptide variants must be synthesised with these in mind (Matsubayashi, 2012; Imin et al. 2013).

A recently reported technology, based on synthetic peptide assays, may assist in overcoming the issue of genetic redundancy that has hindered elucidation of peptide function by loss of function mutants. It was shown that by substituting Gly6 in the CLV3 peptide domain for Ala or Thr, dominant-negative clv3 phenotypes were obtained (Song et al., 2013). When applied in combination with the unsubstituted CLV3 peptide, it was shown that the antagonistic effect was a result of competition between the two peptides. It was hypothesised that CLV3Thr6 _{was able to bind the CLV3}

receptor without eliciting a response. This concept was also used to make antagonists for CLE8 and CLE22 peptides (Song et al., 2013). However, since this method was first described, it has been found that similar dominant-negative effects cannot be

achieved by substituting amino acids of other peptides, such as other CLEs or IDA peptides Czyzewicz et al. (2015).

Additionally, methods such as targeted in silico analyses, mutant analyses and transcriptomic analyses, with careful experimental design, can be used to assist with gene identification and functional elucidation.

These tools and strategies were used to devise a project investigating the CEP family in

Arabidopsis. Preliminary work indicated that members of the CEP family were induced

by environmental cues in Arabidopsis and legume species (Radzman, Imin and

Djordjevic, 2012, personal communication; Imin et al. 2013). Therefore, the overall aim of this thesis was to increase our understanding of the role(s) of Arabidopsis CEPs in modulating root development in response to environmental cues. To initate this study, the CEP family was investigated in more detail (Chapter 3). The relationships between

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CEP peptides were studied, their induction by environemtnal cues were explored and the effects of over-presenting CEP peptides were examined. To uncover specific mechanisms of CEP action, CEP3 was studied in more detail. In Chapter 4, a cep3 mutant was isolated and characterised. This mutant, together with other genetic tools, was used to pinpoint the role of CEP3 in root development. Finally, in Chapter 5, the role of CEP3 in nutrient pathways was explored using transcriptomics. This project has contributed significanly to our understanding of root development in the context of nutritional regulation and has provided a wealth of information about and tools for the CEP family.