Plants have evolved various defense strategies to counter the constant threat from predators and infectious organisms, including physical deterrents such as thorns, sap, and trichomes, alongside a range of secondary metabolites antagonistic to threatening organisms.
Cyclotides are a unique family of plant defense proteins that are structurally defined by their macrocyclic peptide backbone and a knotted arrangement of three disulfide bonds, the combination of which is known as a cyclic cystine knot motif. This framework promotes their extraordinary stability against degradation at high temperatures, extremes of pH, and in the presence of otherwise proteolytic enzymes. Having thus far been described in relatively few plant species, interest in the discovery of new cyclotides has been driven by their numerous plant defense-associated bioactivities along with a host of pharmaceutically relevant bioactivities. Being tolerant to amino acid substitution exclusive of the residues comprising their cystine knot core, the cyclotide framework has also been exploited as a stable drug design scaffold for the incorporation of bioactive peptide epitopes with short half-lives in vivo.
Possessing similar ethnobotanical significance to previously investigated cyclotide-containing plants, and given literature reports describing cyclotide-like activities within its extracts, the fabaceous species Clitoria ternatea L. (butterfly pea) was selected for investigation as a possible source of novel cyclotides. The first section of this thesis describes the mass spectrometric characterisation of the first cyclotides within a Fabaceae plant species. Upon comparison with previously described cyclotides, some of the novel cyclotides were found to possess unusual sequence motifs at locations considered important to their biosynthesis in other plants. Given these results, the second approach of the thesis concerned the elucidation of cyclotide-encoding genes in the butterfly pea. This study revealed the unusual nature of their biosynthetic origin to be a cyclotide-encoding domain embedded within Fabaceae albumin-1 in place of a knottin domain typically observed in other Fabaceae plants. Due to the close structural similarities and insecticidal activities of the Fabaceae cyclotide and a well characterised Fabaceae albumin-1 knottin, the interpretation of this thesis is that cyclotides may have evolved in Fabaceae via divergent evolution from a linear precursor such as the displaced albumin-1 knottin.
Another approach to cyclotide discovery taken in this thesis involved the searching of publicly accessible EST (expressed sequence tag) databases to find candidate cyclotide-encoding nucleotide sequences. Following the discovery of matches to a Fabaceae cyclotide sequence query within Petunia accessions, proteomic and genetic evaluation of Petunia x hybrida revealed the first evidence of cyclotides as well as acyclotides within Solanaceae. The genes encoding petunia cyclotides were found to have features that distinguish them from previously characterised Rubiaceae, Violaceae as well as Fabaceae cyclotide genes, which suggests that cyclotides may exist in many more plant families and be the products of diverse genetic architectures.
This thesis further explores the intra-tissue spatial distribution of cyclotides within a petunia leaf sample, using MALDI imaging. The highest concentrations of cyclotide signals were found to co-localise with the vascular features of the leaf, a result which may be suggestive of a defense role for Solanaceae cyclotides given a similar pattern of defense molecule presentation in the literature for Arabidopsis (Brassicaceae family).
Given the unique analytical challenges posed by cyclotides which limit their rate of discovery in proteomic studies, this thesis examines the effects of orthogonal separation and chemical derivatisation strategies in efforts to improve the rate of cyclotide discovery. Optimisation of the orthogonal pre-fractionation strategy resulted in the detection of an order of magnitude greater number putative cyclotide analytes within the test sample, and the N-terminal derivatisation of the linearised test cyclotide with an Edman reagent resulted in significant improvements in peptide fragmentation.
Overall, these findings provide crucial insights into the genetic origins of cyclotides, demonstrate the non-uniform intra-tissue localisation of cyclotides, and provide a basis for their expedited discovery via proteomic approaches.