a, HPLC chromatogram of purified, refolded Stomagen. Peaks 1 and 2 in UV chromatogram were collected and subjected to MALDI-TOF mass spectrometry (b, d) as well as for bioassays (c, e). b, MALDI-TOF spectrum of peak 1 from a. A single-charged peptide corresponding to synthetic Stomagen peptide was observed at m/z = 5,118.5 ([M+H]+) and a double charged peptide at m/z = 2,559.8 ([M+2H]2+). c, Confocal image of cotyledon epidermis from wild-type seedling grown a solution with peak 1. Severe stomatal clustering and overproduction of stomata are observed. Scale bar, 20 μm. n = 8. d, MALDI-TOF spectrum of peak 2 from a. e, Confocal image of cotyledon epidermis from wild-type seedling grown in a solution with peak 2 from a, with no stomatal clustering, indicating that the fraction is not bioactive. Scale bar, 20 μm. n = 6. f, HPLC chromatogram and bioassays of an independent batch of Stomagen peptides used for QCM analysis in direct comparison with non-folding mutant Stomagen peptides in Fig. 3c. Peaks 1 and 2 in UV chromatogram were collected and subjected for bioassays. Insets: confocal microscopy images of cotyledon epidermis from wild-type seedling grown a solution with peak 1 (bioactive) and peak 2 (non-active) for 5 days. Scale bars, 50 μm. n = 8 (peak 1); n = 6 (peak 2). g, HPLC chromatogram of purified, mutant Stomagen peptide in which all cysteine residues were substituted to serine residues (Stomagen_6C→S). The mutant Stomagen peptide yielded a single peak, which was subjected for bioassays followed by confocal microscopy (inset). No stomatal clustering was observed, indicating that non-folding Stomagen peptide is not bioactive, confirming the previous results18. Scale bar, 50 μm. n = 8 for each peptide treatment.