Extended Data Figure 4. Numbers of stomatal and non-stomatal cells in WT, epf2-1, epf2-2 and crsp-1 mutants at the elevated CO₂ concentration, and mutations in the negative-regulatory extracellular signals of stomatal development.

The secreted EPF signalling pro-peptides have been identified as extracellular pro-peptide ligands that mediate the repression of stomatal development via extracellular signalling^{7–9,22–24,27}. Abaxial cell densities for stomatal cells (a) and non-stomatal cells (b; all epidermal pavement and SLGC cells except guard cells) (per mm²) in mature cotyledons (10 DAG) of WT, epf2-1, epf2-2 and crsp-1 mutants grown at 500 p.p.m. CO₂. Note that the stomatal density effects in epf2 mutants are larger than those on stomatal index (see the main text, Methods and Extended Data Fig. 1c legend). *, P<0.05 for comparisons with WT. c, d, Seedlings carrying mutations in the negative-regulatory extracellular signals of stomatal development, EPF1 and CHALLAH (EPFL6), did not exhibit inverted CO₂ control of stomatal development in cotyledons. Stomatal indices of 10-day-old WT (Col), epf1-1 single mutant⁷ (c) and challah single mutant²¹ (d) seedlings grown at low (150 p.p.m.) and elevated (500 p.p.m.) CO₂ concentrations are shown. In all panels, error bars, mean ± s.e.m., n = 20. *, P<0.05, using ANOVA and Tukey’s post-hoc test.