Butterfly Pea (Clitoria ternatea), a Cyclotide-Bearing Plant With Applications in Agriculture and Medicine

Abstract

The perennial leguminous herb Clitoria ternatea (butterfly pea) has attracted significant interest based on its agricultural and medical applications, which range from use as a fodder and nitrogen fixing crop, to applications in food coloring and cosmetics, traditional medicine and as a source of an eco-friendly insecticide. In this article we provide a broad multidisciplinary review that includes descriptions of the physical appearance, distribution, taxonomy, habitat, growth and propagation, phytochemical composition and applications of this plant. Notable amongst its repertoire of chemical components are anthocyanins which give C. ternatea flowers their characteristic blue color, and cyclotides, ultra-stable macrocyclic peptides that are present in all tissues of this plant. The latter are potent insecticidal molecules and are implicated as the bioactive agents in a plant extract used commercially as an insecticide. We include a description of the genetic origin of these peptides, which interestingly involve the co-option of an ancestral albumin gene to produce the cyclotide precursor protein. The biosynthesis step in which the cyclic peptide backbone is formed involves an asparaginyl endopeptidase, of which in C. ternatea is known as butelase-1. This enzyme is highly efficient in peptide ligation and has been the focus of many recent studies on peptide ligation and cyclization for biotechnological applications. The article concludes with some suggestions for future studies on this plant, including the need to explore possible synergies between the various peptidic and non-peptidic phytochemicals.

Keywords: anthocyanins; butelase; forage crop; medicinal plant; organic pesticide; peptides.

FIGURE 1

Timeline of the key studies and milestones on Clitoria ternatea research from the 1950s to the present. The biological (blue) and biochemical (purple) studies pursued from the 1950s to early 1970s characterized the properties of roots and seeds. Toward the end of the 1970s, researchers began to isolate and characterize the phytochemical compounds from C. ternatea. Ternatins, the anthocyanins that render C. ternatea its vivid blue color, were first isolated in 1985; and the structure of the largest of the ternatins, ternatin A1, was characterized in 1989. Further isolation and characterization of the ternatins in C. ternatea led to the elucidation of the ternatin biosynthetic pathway in 1998. Parallel to the studies that characterized the phytochemical composition of C. ternatea, were agricultural studies that evaluated C. ternatea as a forage and fodder crop. A series of field studies in Queensland, Australia lead the development and eventual release of the C. ternatea Milgarra cultivar in 1991. From 2001 to the present, studies have been determining the pharmacological activities and biological activities of C. ternatea extracts. In 2011, cyclotides, the circular insecticidal molecules which can also be used as scaffolds for peptide-based therapeutics, were discovered in C. ternatea. While cyclotides had previously been characterized in other angiosperm species, C. ternatea is to date, the only legume that is known to produce them. In 2014, butelase-1, the ligase that facilitates cyclization in C. ternatea cyclotides, was discovered and characterized. Cyclotides and the auxiliary enzymes, have applications both in modern medicine and agriculture. In 2017, Sero-X^® an eco-friendly insecticide made from C. ternatea extracts was registered for commercial use in Australia.

FIGURE 2

Clitoria ternatea (A) flower, (B) pods, (C) leaves, and (D) roots with nodules. The C. ternatea flower consists of the stamen (st), pistil (p), sepals (sp), and corollae. The corollae consist of five petals: one banner (b), two wings (w) and two keels (k). C. ternatea has pinnate compound leaves, flat and pointed pods and roots that produce nodules (n).

FIGURE 3

Distribution of Clitoria subgenus Clitoria species adapted from Fantz, 1977. Points of occurrence are approximate. Map data from Openstreetmap.org. Symbols represent: ◼ C. biflora, □ C. heterophylla, Δ C. kaessneri, • C. lasciva, + C. ternatea.

FIGURE 4

Proposed ternatin biosynthetic pathway. Adapted from Terahara et al. (1998). Beginning with ternatin C5 (PubChem CID 10843319), ternatin A1 (PubChem CID 16173494) can be produced through the addition of four p-coumaroyl (C) and four glucosyl moieties (G) at the 3′ sidechain (in blue) and 5′ sidechain (in orange). The other ternatins are products of the intermediate steps.

FIGURE 5

Graphical representations of C. ternatea cyclotide structure and diversity. (A) Topological depiction of Cter M displaying the position and threading of disulfide bonds. (B) Diversity of residues at non-cystine positions of 74 sequence defined C. ternatea cyclotides. (C) Loop length diversity and disulfide connectivity map of C. ternatea cyclotides.

FIGURE 6

Structure of a Fabaceae albumin-1 cyclotide prepropeptide exemplified by Cter M. The linear Cter M precursor consists of the endoplasmic reticulum (ER) signal (red), the cyclotide domain (blue) which replaces the typical Fabaceae albumin-1 b-chain, the a-chain (yellow) and the C-terminal interlinker region (green). A specialized asparaginyl endopeptidease (AEP, butelase-1 (PDB code: 6DHI) effects head-to-tail cyclization of the Cter M domain of the precursor in planta and results in a mature CterM cyclotide (PDB code: 2LAM).