Distribution and Evolution of Circular Miniproteins in Flowering Plants

Abstract

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids (;125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

Discovery of Linear Cyclotides in Monocot Plant Panicum laxum of Poaceae Family Provides New Insights into Evolution and Distribution of Cyclotides in Plants

“…Similar to cyclotide genes of the Violaceae family, they do not contain an intron in the ER signal region (38). The ER signal sequence is likely to play a role in directing the protein into the ER lumen for oxidative folding and subsequently entering the secretory pathway.…”

Micropropagation of Viola uliginosa (Violaceae) for endangered species conservation and for somaclonal variation-enhanced cyclotide biosynthesis

“…After reduction and alkylation, the differences in mass corresponded to the presence of three disulfide bonds, and Glu-C cleavage resulted in one linear fragment (mass difference corresponding to one H 2 O), fulfilling the criteria described by Gruber et al (2008) to classify them as cyclotides. Fifteen regenerated plants, including 9 diploids (2x) and 6 tetraploids (4x) were suitable for analysis of relative cyclotide biosynthesis.…”

“…The methods used for cyclotide isolation, HPLC purification, cysteine reduction and alkylation as well as trypsin and/or Glu-C cleavage as criteria for cyclotide identification were as described by Gruber et al (2008).…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…By contrast, <10% of the species from the Rubiaceae family that have so far been investigated contain cyclotides. [29] An individual plant typically contains a suite of many homologous cyclotides. For example, a study of the native N. L. Daly et al…”