The key drivers of cyclotide production in butterfly pea

GK Oguis, EK Gilding, A Poth, MA Jackson and DJ Craik

Institute for Molecular Bioscience, University of Queensland, St Lucia, 4072, Australia

Butterfly pea (Clitoria ternatea) produces interesting disulfide rich head-to-tail cyclized peptides, called cyclotides. Butterfly pea is the first and only reported cyclotide-producing species from the Fabaceae family, an economically important family that includes vital crops such as soybeans and peanuts. Cyclotides are hypothesized to have evolved as means for plants to defend themselves from biotic factors. Their defensive powers are useful in the agricultural sector where a commercial butterfly pea product consisting of extracts containing cyclotides will soon be on the market. The cyclotides in butterfly pea are biosynthesised from precursors embedded within members of the albumin-1 multi-gene family. Although the pathway that matures cyclotides from these precursors is known from in vitro experiments, the factors that control the rates of cyclotide production in planta are unknown. Why they are constitutively produced in such great abundance is also not well understood. To shed light on these questions, we determined the cyclotide expression profiles of 150 butterfly pea plants at the peptide level. Here we show that different individuals variably express their cyclotide compliment. The major cyclotide of vegetative tissue, CterM, was found to be missing in a small number of seed-grown plants. The amino acid sequences of the CterM-negative plants contain non-silent mutations in their full length precursor albumin sequence, and these polymorphisms may contribute to the lack of mature CterM expression. Furthermore, promoter regions of the accessions in which CterM is either highly expressed or under-expressed are investigated to determine the potential regulatory elements that drive cyclotide expression. Together our evidence suggests butterfly pea cyclotides constitute a part of butterfly pea's innate defences.