Figure 2. EPF2 expression is regulated by CO₂ concentration and is essential for CO₂ control of stomatal development.

a, EPF2 messenger RNA levels in developing 5 DAG (days after germination) cotyledons of WT and ca1 ca4 seedlings, showing induction, at the elevated CO₂ concentration in the WT but not ca1 ca4. Levels were normalized to those of the CLATHRIN gene. The insets show the normalized RNA-seq expression of EPF2 exons from an RNA-seq experiment (5 DAG). b–d, MUTE expression correlates with the stomatal development phenotype of the ca1 ca4 mutant. Confocal images showing MUTEpro::nucGFP expression (green) in developing (5 DAG) cotyledons of WT and ca1 ca4 plants (b). Scale bars, 100 µm. Quantitation of MUTEpro::nucGFP-expressing cells in the WT and two independent lines in the ca1 ca4 background, at low and elevated CO₂ concentrations (c). d, Stomatal index in WT plants and plants carrying either of two independent mutant alleles of epf2, at low and elevated CO₂ concentrations, demonstrating that epf2 mutants show an inversion of the elevated CO₂-mediated control of stomatal development. Error bars, mean ± s.e.m., n = 10 in a and n = 20 in c and d. ***, P<0.00005; **, P<0.005; *, P<0.05, using ANOVA and Tukey’s post-hoc test.