Extended Data Fig. 9: The catalytic activity of EPP5 and EPP6 is essential for their functions.
From: Subtilase-mediated maturation of EPF1 and EPF2 is crucial for stomatal patterning
a, Schematic diagrams of the various domains of the EPP5 and EPP6 proteins. D, H, and S are their catalytic triad. b–i, Representative confocal microscopy images of cotyledon abaxial epidermis from 5-day-old seedlings of wild type (b), epp1/2/3/4/5/6 (c), pEPP5::gEPP5-YFP in epp1/2/3/4/5/6 (d), pEPP6::gEPP6-YFP in epp1/2/3/4/5/6 (e), pEPP5::gEPP5D-A-YFP in epp1/2/3/4/5/6 (f), pEPP5::gEPP5DHS-AAA-YFP in epp1/2/3/4/5/6 (g), pEPP6::gEPP6D-A-YFP in epp1/2/3/4/5/6 (h), and pEPP6::gEPP6DHS-AAA-YFP in epp1/2/3/4/5/6 (i). Images were taken under the same magnification. Scale bar, 20 µm. j, Quantification of the stomatal number of indicated genotypes. k, Quantification of the stomatal index of indicated genotypes. For box plots (j, k), box limits indicate 25th and 75th percentiles; central lines indicate medians; whiskers display minimum and maximum values. Dots represent individual measurements per group. Different lowercase letters indicate statistically significant differences based on one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05). The exact P values for all comparisons are provided in Supplementary Table 2. Sample sizes (number of biological replicates, each representing a cotyledon of an independent seedling): n = 17 (wild type), 8 (epp1/2/3/4/5/6), 8 (pEPP5::gEPP5-YFP in epp1/2/3/4/5/6), 10 (pEPP6::gEPP6-YFP in epp1/2/3/4/5/6), 8 (pEPP5::gEPP5D-A-YFP in epp1/2/3/4/5/6), 12 (pEPP5::gEPP5DHS-AAA-YFP in epp1/2/3/4/5/6), 9 (pEPP6::gEPP6D-A-YFP in epp1/2/3/4/5/6), 9 (pEPP6::gEPP6DHS-AAA-YFP in epp1/2/3/4/5/6). l, Western blot using GFP antibody recognizing the EPP5–YFP, EPP5D-A–YFP, EPP5DHS-AAA–YFP, EPP6–YFP, EPP6D-A–YFP, and EPP6DHS-AAA–YFP in stable transgenic Arabidopsis seedlings. The positions of the zymogen that still retain the inhibitor I9 domain are indicated by a black asterisk, while the mature processed forms of EPP5 and EPP6 are indicated by a red asterisk.
