Skip to main content
. 2021 Oct 27;34(1):455–476. doi: 10.1093/plcell/koab260

Figure 5.

Figure 5

Expressing the cutinase gene CDEF1 decreases stomatal cluster formation in ectopic MYB16 lines. A, A diagram showing the biosynthesis of one type of cutin monomer. The C16/C18 fatty acid from plastids is transformed into the hydroxylated acyl-CoA intermediate and then becomes cutin monomers in the ER. The transport of the monomers supplies the material required for the polymerization of the cuticle layer outside the cell walls. The polymerization of the cuticle layer is mediated by cutin synthase. Genes involved in the reaction are labeled in blue. B, Relative mRNA expression of cuticle biosynthesis genes in ectopic MYB16 lines. A 7-dpg WT is used as a reference (equal to 1). *P < 0.05; **P < 0.01; ***P < 0.001, by Student’s t test. Data are mean (sd). Three biological replicates showed similar results. C–H, TEM images of the lower epidermis from 7-dpg true leaves of the WT (C) and (F), BASLp:MYB16-YFP (D) and (G), and CYP77A6p:MYB16-VP16 (E) and (H). Both pavement cells (C)–(E) and stomatal-lineage cells (F)–(H) showed thicker cuticle deposition (white brackets) on the cell surface in BASLp and CYP77A6p lines than the WT. I–L, Quantification of cuticle (I) and (J) and cell wall (K) and (L) thickness from (C) to (H). The cuticle thickness of epidermal (I) and (K) and stomatal lineage cells (J) and (L) increased in ectopic MYB16 lines compared to the WT, but cell wall thickness decreased. Data were obtained from 200 to 300 regions of lower epidermal cells. *P < 0.05, by Student’s t test. Data are medians (interquartile range). Gray dots represent Tukey outliers. M, Quantification of penetrated TB showing that MYB16-SRDX plants are more permeable than BASLp or CYP77A6p plants. After ectopically introducing the cutinase gene CDEF1 into BASLp or CYP77A6p lines, seedlings showed more penetrated TB compared with the control in each background. The TB absorbance (A626) is normalized by chlorophyll absorbance (A430). P < 0.05. Kruskal–Wallis with Dunn’s test. Data are means (se). N, O, Quantification of stomatal density (N) and stomatal index (O) shows the partial rescue of MYB16 ectopic expression plants by ectopically expressing the cutinase gene CDEF1. n = 20 10-dpg seedlings. *P < 0.05, by Wilcoxon signed-rank test. Data are medians (interquartile range). P, Q, The number of stomatal clusters (P) and cluster frequency (Q) decreased after ectopically expressing the cutinase gene CDEF1. For (P), the values were obtained from the sum of the events in a total of 20 lower-epidermis samples. Rescue percentage is the difference in the number of cluster events between the control and ML1p:CDEF1-flag divided by the event number in the control. For (Q), cluster frequency is the cluster event number divided by the number of stomatal groups. P < 0.05. One-way ANOVA with Tukey post hoc test. Data are means (se). For (C)–(H), shared scale bars, 200 nm, in (C) and (F). For (M)–(Q), two biological replicates and two individual lines in each background showed similar results. See also Supplemental Figures S8–S10.