Cyclotides associate with leaf vasculature and are the products of a novel precursor in petunia (Solanaceae)

Abstract

Cyclotides are a large family of plant peptides that are structurally defined by their cyclic backbone and a trifecta of disulfide bonds, collectively known as the cyclic cystine knot (CCK) motif. Structurally similar cyclotides have been isolated from plants within the Rubiaceae, Violaceae, and Fabaceae families and share the CCK motif with trypsin-inhibitory knottins from a plant in the Cucurbitaceae family. Cyclotides have previously been reported to be encoded by dedicated genes or as a domain within a knottin-encoding PA1-albumin-like gene. Here we report the discovery of cyclotides and related non-cyclic peptides we called "acyclotides" from petunia of the agronomically important Solanaceae plant family. Transcripts for petunia cyclotides and acyclotides encode the shortest known cyclotide precursors. Despite having a different precursor structure, their sequences suggest that petunia cyclotides mature via the same biosynthetic route as other cyclotides. We assessed the spatial distribution of cyclotides within a petunia leaf section by MALDI imaging and observed that the major cyclotide component Phyb A was non-uniformly distributed. Dissected leaf midvein extracts contained significantly higher concentrations of this cyclotide compared with the lamina and outer margins of leaves. This is the third distinct type of cyclotide precursor, and Solanaceae is the fourth phylogenetically disparate plant family to produce these structurally conserved cyclopeptides, suggesting either convergent evolution upon the CCK structure or movement of cyclotide-encoding sequences within the plant kingdom.

FIGURE 1.

BOXSHADE alignment of cyclotide precursors in genus Petunia. A, PETUNITIDE sequences. B, consensus sequences derived from matched ESTs. Asterisks at the C termini of sequences indicate stop codons. Red bar denotes predicted signal motifs, and green bars denote cyclotide or acyclotide domains. Triangles denote prototerminal amino acids of encoded cyclotides. Disulfide connectivity is based upon previously characterized cyclotides. Sequences translated from EST data only are listed in supplemental Table S1 (note paired clones (F + R)).

FIGURE 2.

MALDI-TOF MS of petunia leaf and root extracts reveals putative cyclotide masses. Following reduction of disulfide bonds, free thiols were alkylated, and peptide backbones were enzymatically digested with endoproteinase Glu-C. A, native leaf extract. B, reduced and carbamidomethylated leaf extract. C, reduced, carbamidomethylated, and enzymatically digested leaf extract. D, native root extract. E, reduced and carbamidomethylated root extract. F, reduced, carbamidomethylated, and enzymatically digested root extract. G, the structures of native, alkylated, and digested Phyb A (peak a) and Phyb M (peak b) are schematically represented beside their corresponding spectra.

FIGURE 3.

MALDI-TOF/TOF analyses of cyclotide and acyclotide species isolated from petunia root. A, tandem MS analysis of the m/z 3435.47 precursor (Phyb A). B, tandem MS analysis of the m/z 3736.52 precursor (Phyb M). C, tandem MS analysis of the m/z 3600.67 precursor (Phyb K). D, tandem MS analysis of the m/z 2978.28 precursor (Phyb M fragment).

FIGURE 4.

Alignment of cylcoviolacin O17 with cyclotide Phyb A (86% homology) and acyclotides Phyb K and Phyb M from P. x hybrida. Disulfide connectivity based upon previously characterized cyclotides is denoted by solid lines between boxed cysteines, and intercysteine loops are indicated. Loop 6 is not present in Phyb K or Phyb M, which are both acyclic.

FIGURE 5.

MALDI-MSI of a petunia leaf. The localizations of four distinct m/z signals are indicated with intensity scales relative to average spectrum maximum inset. A, average mass spectrum for the data acquired from the leaf section. B, dark field microscope image of a paradermal (adaxial longitudinal) cryosection of P. x hybrida leaf. C, localization of m/z 3069. D, localization of m/z 3110. E, localization of m/z 3426. F, localization of m/z 3463. G, overlay of m/z 3426 (green) and m/z 3069 (red) signals.

FIGURE 6.

Relative quantitation of Phyb A across petunia leaf regions. A, representative petunia leaf highlighting the regions sampled for LC/MS analysis. Green highlight, midvein; red highlight, lamina; blue highlight, leaf periphery. B, relative extracted ion chromatogram peak intensities for signal at 1535.0²⁺ in LC/MS analyses (corresponds to m/z 3069 in MALDI experiments) of boiled plant extracts. Error bars, S.E. (n = 10). Asterisks denote significant differences at 0.001 < p < 0.01 (**) or 0.01 < p < 0.05 (*) following one-way analysis of variance using Bonferroni's multiple comparison test. Scale bar, 1 cm.

FIGURE 7.

Comparison of prototypic cyclotide and acyclotide-encoding gene structures in angiosperms. A, variation in cyclotide and related knottin gene architectures among angiosperms. B, acyclotide and knottin domains. C, cyclotide domains. Signal sequence is shown in white boxes. Knottin and acyclotide domains are shown in light blue boxes. Cyclotide domains are shown in orange boxes. N-terminal pro-domains are shown in green boxes. C-terminal repeats are shown in mauve boxes. V, Violaceae; R, Rubiaceae; P, Poaceae; F, Fabaceae; C, Cucurbitaceae; S, Solanaceae.