a–j, Representative confocal images of cotyledon abaxial epidermis from 10-day-old light-grown transgenic seedlings of the following genotypes, each carrying Est::STOMAGEN construct: epf2 (a, b); dominant-negative ER (ER(ΔK)) in er (c, d); epf1 (e, f); dominant-negative ERL1 (ERL1(ΔK)) in erl1 (g, h); er erl1 erl2 (i, j). For each genotype, a control uninduced phenotype (a, c, e, g, i) and induced STOMAGEN overexpression (iSTOMAGEN) (b, d, f, h, j) are shown. Blocking ER or lacking EPF2 produces small stomatal-lineage cells due to excessive entry divisions (a, c; yellow brackets). iSTOMAGEN confers stomatal clusters and small stomatal-lineage cells are no longer present (b and d). Blocking ERL1 or lacking EPF1 causes a stomatal pairing due to a violation of one-cell-spacing rule (e, g; dots). iSTOMAGEN enhances stomatal cluster phenotype in these genotypes (f, h). iSTOMAGEN does not enhance stomatal clustering defects in er erl1 erl2 (i, j). Images were taken under the same magnification. Scale bars, 30 µm. n = 29 (a); n = 24 (b); n = 16 (c); n = 17 (d); n = 22 (e); n = 23 (f); n = 17 (g); n = 20 (h); n = 24 (i); n = 24 (j). k–m, Stomatal density (number of stomata per mm2) (k); stomatal index (% of number of stomata per stomata + non-stomatal epidermal cells) (l); and stomatal cluster distribution (in %) (m) from 10-day-old abaxial cotyledons of transgenic lines of each genotype carrying Est::STOMAGEN construct. −, no induction; +, induced by 10 µM oestradiol. Stomagen overexpression significantly increases stomatal density in all genotypes except for er erl1 erl2 and tmm. Error bars indicate s.e.m. ***P < 0.001; **P < 0.01; NS, not significant; Welch 2-sample t-test. Number of seedlings subjected to analysis, n = 14–16. Total numbers of stomata counted: wt, no induction 1,277, induction 2,639; epf1, no induction 1,390, induction 3,485; ERL1(ΔK) erl1, no induction 1,573, induction 3,991; epf2, no induction 2,502, induction 3,317; ER(ΔK) er, no induction 2,899, induction 4,397; tmm, no induction 2,948, induction 3,212; er erl1 erl2, no induction 4,454, induction 4,464. All genotypes carry Est::STOMAGEN. wt, no induction n = 16, induction n = 14; epf1, no induction n = 16, induction n = 17; ERL1(ΔK) erl1, no induction n = 15, induction n = 15; epf2, no induction n = 15, induction n = 15; ER(ΔK) er, no induction n = 15, induction n = 15; tmm, no induction n = 15, induction n = 15; er erl1 erl2, no induction n = 15, induction n = 15.