Investigating the diversity of CLE proteins and their function in Arabidopsis thaliana

Our understanding of the importance of peptide hormone signalling in the growth and development of plants has improved in recent years with the identification of several protein families that regulate cell division and differentiation within a variety of meristematic tissues. One family in particular, known as CLAVATA3/ESR-RELATED (CLE) proteins, has been shown to be a diverse functioning family controlling differentiation of the xylem and phloem from the cambium and affecting root development. Previously CLE proteins were thought to contain a single CLE peptide at the C-terminal end however through bioinformatic investigation using a combination of BLAST and MEME FIMO we have identified numerous proteins spread across the plant kingdom that contain between two and twenty-four CLE peptides in a tandem array separated by a conserved spacer region. These Multi-CLE peptide proteins have been identified across a variety of agriculturally important crop species but are not present in species such as Arabidopsis thaliana. Due to the radial growth of the hypocotyl Arabidopsis thalaiana is an ideal model organism for investigating vascular development as its simple development allows for easy identification of developmental changes. Expression of the Gossypium raimondii Multi-CLE peptide protein in A. thaliana alters vascular tissue organisation in the hypocotyl, indicating that the protein is functional in vivo. Presumably protein cleavage occurs to release the individual CLE peptide(s) which are then recognised by native receptors and downstream effectors are activated or inhibited to alter cell division and differentiation. Western blot analysis suggests that peptides and subsequent spacers are cleaved off from the protein together, however further experimental analysis is required to elucidate this. The application of synthetic peptides indicates that the peptides are predominantly categorised as A-Type peptides as they are shown to be affecting root growth. In addition to this, we have identified that the spacer region appears to have no effect on root growth but also does not impede the activity of the peptide insinuating spacers at the N-terminal end of a peptide are cleaved off. However, we have yet to elucidate whether spacers at the C-terminal end affect peptide activity and whether they two are cleaved off through another proteolytic process.