Volume 9, Issue 6, Pages 926-938 (June 2016) Dissecting Abscisic Acid Signaling Pathways Involved in Cuticle Formation  Fuqiang Cui, Mikael Brosché, Mikko T. Lehtonen, Ali Amiryousefi, Enjun Xu, Matleena Punkkinen, Jari P.T. Valkonen, Hiroaki Fujii, Kirk Overmyer  Molecular Plant  Volume 9, Issue 6, Pages 926-938 (June 2016) DOI: 10.1016/j.molp.2016.04.001 Copyright © 2016 The Author Terms and Conditions

Figure 1 Summary of Known Pathways Regulating Cuticle Formation. Some environmental factors and transcription factors are known to regulate cuticle biosynthesis. However, the relationships between these pathways and the regulation of these transcription factors remain undefined (dashed lines). Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions

Figure 2 Core ABA Signaling Regulates Cuticle Permeability. (A) Dye exclusion assay. Fully elongated leaves of 3-week-old plants were stained with 5 μl of 0.05% toluidine blue (TB) drops for 2 h. Leaves with deficient cuticle stained dark blue. Scale bar, 0.5 cm. (B) TB-stained areas were quantified in ImageJ. Combined results of four experiments (n = 12 in each independent biological repeat) were analyzed in a linear mixed model with single-step p-value adjustment. Error bars represent SE of means (N = 48 in total). Letters above the bars indicate significance groups (p < 0.05). (C) Young leaves were not TB permeable in snrk2.236. Symptoms of whole rosettes of 2-week-old plants immersed in TB solution for 30 min. Scale bar, 1 cm. Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions

Figure 3 Core ABA Signaling Regulates Cuticle Formation via Modulation Cuticle-Related Gene Expression. Gene expression in 3-week-old plants (whole rosettes) was examined with real-time quantitative RT–PCR (qPCR). Expression of various cuticle-related genes (transcription factors, biosynthesis enzymes, transporters) were monitored in four independent biological repeats. Combined means of expression value (log10) from all experiments were clustered with the R function heatmap.2. Stars indicate significant difference to Col-0 (t-test: ***p < 0.001; **p < 0.01; *p < 0.05). Gene classifications are indicated with white-black (Pathway) and color (Function) to present their molecular roles in cuticle formation. For the raw data used in creating this heatmap, see Supplemental Table 1. Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions

Figure 4 Humidity-Responsive Changes in Cuticle Formation Are Independent of ABA Signaling. (A) Low-humidity treatment decreased cuticle permeability. Three-week-old plants grown under 100% humidity (white bars) or shifted into 70% humidity for 1 week (gray bars) were stained with TB. Scale bar, 0.5 cm. Combined results of three experiments (n = 12 in each independent biological repeat) were analyzed in a linear mixed model with single-step p-value adjustment. Error bars represent SE of means (N = 36 in total). Letters above the bars indicate significance groups (p < 0.05). (B) Changes in humidity-regulated expression of cuticle genes independent of SnRK2s. Three-week-old plants grown under 100% humidity or treated with 3 h of 70% humidity were examined with qPCR. Means of three repeats were clustered into a heatmap. A manual adjustment was subsequently applied to order the presentation according to 100% and 3-h 70% humidity treatments. Gene classifications are indicated with white-black (Pathway) and color (Function) to present their molecular roles in cuticle formation. Stars indicate significant differences between the two humidity treatments (t-test: *p < 0.05; **p < 0.01; ***p < 0.001). For the raw data used in creating this heatmap, see Supplemental Table 2. Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions

Figure 5 Cuticle Function in Humidity Adaptation. (A) Arabidopsis mutants with permeable cuticle are more adapted to water-saturated conditions. Well-watered plants were covered with a lid to maintain 100% humidity. Representative pictures of 4-week-old plants are shown. Control, normal watering and constant 70% humidity; Saturated, well watered at 100% humidity. Scale bar, 1 cm. Red arrows indicate water-soaked leaves. (B) Similarity analysis of Arabidopsis proteins involved in cuticle formation. The color key (right) shows the percentage identity ranging from 27 (lowest) to 100 (highest). The heatmap is ordered based on the increasing average percent identity from 60 cuticle genes with the highest hits belonging to Arabidopsis (rightmost column). The tree is clustered based on the correlation matrix between columns with Euclidean distance measure. Gene classifications are indicated with white-black (Pathway) and color (Function) to present their molecular roles in cuticle formation. Lyco., lycophyte. For the raw data used in creating this heatmap, see Supplemental Table 3. (C) Exogenous ABA suppressed expression of cuticle-related genes in Physcomitrella. Shoots of 2-month-old Physcomitrella were treated with the indicated ABA concentrations. Gene expression was examined with qPCR. Means and SD of three repeats are shown. Stars indicate significant differences between mock (0 μM) and other ABA concentrations (t-test: *p < 0.05; **p < 0.01; ***p < 0.001). Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions

Figure 6 Regulation of ABA and Cuticle-Related Genes and Botrytis Infection of ABA Mutants. (A) Expression analysis of cuticle- and ABA-related genes in the indicated experiments. Clustering was applied with fold changes (log2) of the indicated experiments. Cuticle-related genes (indicated with a blue box) were collected from TAIR (https://www.arabidopsis.org) and publications; ABA-related genes (indicated with a yellow box) were from Hauser et al. (2011). Genes are separated into four clusters vertically (I, II, III, and IV) and experiments into five groups horizontally (A, B, C, D, and E). Yellow and blue boxes on the right side indicate ABA- and cuticle-related genes, respectively. For the raw data used in creating this heatmap, see Supplemental Table 4. (B and C) Mutants with impaired ABA signaling and permeable cuticle were resistant to Botrytis (B), while ABA mutants with normal cuticles exhibited wild-type immunity to Botrytis (C). Lesion diameter of 4-week-old plants after Botrytis infection (2 × 106 spores ml−1) were measured in ImageJ. Four independent experiments (n = 15 in each independent biological repeat) were combined and analyzed using a linear mixed model with single-step p-value adjustment. Letters above the bars indicate significance groups (p < 0.05). Error bars represent SE of means (N = 60 in total). Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions

Figure 7 Signaling Pathways Regulating Cuticle Formation. (A) Core ABA signaling regulated both cutin and wax formation (black lines) in plants, which was independent but intertwined with humidity-induced signaling (gray lines) and possibly also pathogen signaling (dashed gray lines). SnRK2.2/3/6 was a node upstream of three parallel pathways: cuticle-related signaling, ABF-regulated ABA signaling (gray), and stomata signaling (gray). (B) ABA and humidity-responsive transcription factors. Both ABA and changes in air humidity suppressed the expression of DEWAX and HDG1, but increased the expression of MYB96. MYB94 was oppositely regulated in response to ABA and humidity changes. MYB16 was regulated only by ABA. Molecular Plant 2016 9, 926-938DOI: (10.1016/j.molp.2016.04.001) Copyright © 2016 The Author Terms and Conditions